Analytical biochemistry最新文献_第2页

Label-Free Electrochemical Biosensing of MDR-TB Genes Using Graphite-ZnO Nanofibers on Glassy Carbon Electrodes. 石墨- zno纳米纤维在玻碳电极上对耐多药结核病基因的无标记电化学生物传感。

IF 2.5 4区生物学

Analytical biochemistry Pub Date : 2026-05-05 DOI: 10.1016/j.ab.2026.116143

Dinesh R Rotake, Jitendra B Zalke, Arpita Parakh, Shubham C Anjankar, Shiv Govind Singh, Ranjana Singh

{"title":"Label-Free Electrochemical Biosensing of MDR-TB Genes Using Graphite-ZnO Nanofibers on Glassy Carbon Electrodes.","authors":"Dinesh R Rotake, Jitendra B Zalke, Arpita Parakh, Shubham C Anjankar, Shiv Govind Singh, Ranjana Singh","doi":"10.1016/j.ab.2026.116143","DOIUrl":"https://doi.org/10.1016/j.ab.2026.116143","url":null,"abstract":"<p><p>The rising global burden of multidrug-resistant tuberculosis (MDR-TB) highlights the urgent need for simple, affordable, and reliable diagnostic technologies. In this work, a low-cost, label-free DNA biosensor is developed using graphite-zinc oxide (Graphite-ZnO) nanofibers fabricated by electrospinning and integrated onto glassy carbon electrodes. The biosensor is specifically designed for the detection of drug-resistance-associated genes of Mycobacterium tuberculosis (MTB), with particular emphasis on the embC gene, which is linked to both active and latent TB infections. Single-stranded oligonucleotide probes targeting the embC gene were carefully designed and synthesized, followed by the optimization of surface immobilization protocols to ensure stable and efficient probe attachment onto the nanofiber-modified electrodes. The synthesized nanofibers were structurally and morphologically characterized at a scale of up to 20 μm, confirming the formation of a uniform and interconnected fibrous network suitable for biosensing applications. Electrochemical evaluation of DNA hybridization events was carried out using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The biosensor demonstrated sensitive detection performance, achieving limits of detection of 0.665 pM mL<sup>-1</sup>, 0.326 pM mL<sup>-1</sup>, and 0.51 pM mL<sup>-1</sup> using CV, DPV, and EIS, respectively. The practical applicability of the proposed biosensor was further validated through hybridization assays using DNA extracted from urine samples, confirming its potential as a non-invasive and point-of-care diagnostic platform for MDR-TB detection.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116143"},"PeriodicalIF":2.5,"publicationDate":"2026-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147832712","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}

引用次数: 0

Rapid and Sensitive Detection of H3 AIV HA1 Protein Using a Quantum Dot-Labeled Immunochromatographic Strip. 量子点标记免疫层析条带快速灵敏检测H3 AIV HA1蛋白

IF 2.5 4区生物学

Analytical biochemistry Pub Date : 2026-05-05 DOI: 10.1016/j.ab.2026.116146

Junhui Li, Yamin Zu, Diqing Cao, Haili Wang, Xiao Liu, Jingming Zhou, Aiping Wang

{"title":"Rapid and Sensitive Detection of H3 AIV HA1 Protein Using a Quantum Dot-Labeled Immunochromatographic Strip.","authors":"Junhui Li, Yamin Zu, Diqing Cao, Haili Wang, Xiao Liu, Jingming Zhou, Aiping Wang","doi":"10.1016/j.ab.2026.116146","DOIUrl":"https://doi.org/10.1016/j.ab.2026.116146","url":null,"abstract":"<p><p>Avian influenza virus (AIV) subtype H3 has evolved into a major zoonotic pathogen and poses a potential threat to public health. Hemagglutinin 1 region (HA1) proteins constitute the globular head region of hemagglutinin proteins, contain receptor-binding domains and esterase structural domains, and are an important molecular basis for antigenic variation in AIV. Therefore, a rapid and highly sensitive assay for the HA1 protein of this viral subtype is essential for effective epidemic control. Using quantum dots (QDs) as the core material, a fast-response fluorescent immunochromatographic test strip was developed in this study for the specific recognition of the HA1 protein. The fluorescent probe required for detection was constructed by covalently binding the QDs to a high-affinity HA1 monoclonal antibody (mAb). The test strip works based on the double antibody sandwich principle and can detect the HA1 protein in as little as 15 minutes. The method was validated with recombinant HA1 protein and the visual limit of detection (LOD) was 15.63 ng/mL. The assay exhibited high specificity, demonstrating no cross-reactivity with other prevalent subtypes of AIV, infectious bronchitis virus (IBV), or infectious bursal disease virus (IBDV). In conclusion, this method combines high sensitivity, high specificity and high timeliness, which can provide an effective idea for the detection and rapid diagnosis of avian influenza.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116146"},"PeriodicalIF":2.5,"publicationDate":"2026-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147832690","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}

引用次数: 0

Important Progress in Antimicrobial Peptide Prediction Research in the Past Five Years. 近五年来抗菌肽预测研究的重要进展。

IF 2.5 4区生物学

Analytical biochemistry Pub Date : 2026-05-04 DOI: 10.1016/j.ab.2026.116141

Yun Zuo, Xuan Liu, Xinyue Shao, Zixuan Guo, Sifan Zhu, Zhiqiang Dai

{"title":"Important Progress in Antimicrobial Peptide Prediction Research in the Past Five Years.","authors":"Yun Zuo, Xuan Liu, Xinyue Shao, Zixuan Guo, Sifan Zhu, Zhiqiang Dai","doi":"10.1016/j.ab.2026.116141","DOIUrl":"https://doi.org/10.1016/j.ab.2026.116141","url":null,"abstract":"<p><p>Antimicrobial peptides (AMPs), as key targets for novel anti-infection therapy, have made their efficient and precise identification technology a critical breakthrough in biomedical research. Currently, the high cost of experimental validation severely restricts the development process of antimicrobial peptides, while computational biology methods demonstrate significant advantages due to their cost-effectiveness and efficiency. This review comprehensively examines antimicrobial peptide database resources, with a focus on analyzing the characteristics and application value of mainstream databases such as APD3, DRAMP, and DBAASP, and systematically summarizes the latest advances in feature encoding techniques in recent years. At the methodological level, we discuss in detail the innovative prediction methods that have emerged in the past five years (2020-2024): from traditional structural feature analysis and machine learning algorithms to cutting-edge deep learning and generative model technologies, providing an in-depth comparison of the predictive performance of various methods on different datasets. Finally, based on current research bottlenecks, we prospectively propose future development trends in antimicrobial peptide prediction research, providing direction for scientific research efforts in related fields.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116141"},"PeriodicalIF":2.5,"publicationDate":"2026-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147832692","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}

引用次数: 0

Gold Nanoparticle-Based Electrophoresis-Free Colorimetric Detection Method for Allele-Specific PCR-SNP Genotyping. 基于金纳米颗粒的等位基因特异性PCR-SNP基因分型无电泳比色检测方法。

IF 2.5 4区生物学

Analytical biochemistry Pub Date : 2026-05-02 DOI: 10.1016/j.ab.2026.116142

Surya Kant Verma, Lal Krishan Kumar, Murli Dhar Mitra, Jitendra Kumar, Prashant Singh, Roshan Mohiddin, Varij Nayan, Dheer Singh, Suneel Kumar Onteru

{"title":"Gold Nanoparticle-Based Electrophoresis-Free Colorimetric Detection Method for Allele-Specific PCR-SNP Genotyping.","authors":"Surya Kant Verma, Lal Krishan Kumar, Murli Dhar Mitra, Jitendra Kumar, Prashant Singh, Roshan Mohiddin, Varij Nayan, Dheer Singh, Suneel Kumar Onteru","doi":"10.1016/j.ab.2026.116142","DOIUrl":"https://doi.org/10.1016/j.ab.2026.116142","url":null,"abstract":"<p><p>Conventional allele specific PCR (AS-PCR) genotyping using gel electrophoresis and ethidium bromide (EtBr) is costly, particularly in developing countries. It also poses health risks to working personnel as it requires specialized equipment and toxic dyes like ethidium bromide. Hence, the present study developed a simple and cost-effective colorimetric genotyping method using gold nanoparticles solution (AuNPs) and unmodified primers. Specifically, 15μl of AuNPs solution was found sufficient for detecting an amplicon in 5μl of PCR product. In this approach, the amplified PCR products appear red while the non-amplified PCR products appear blue with a PCR mastermix without a dye. Transmission Electron Microscopy (TEM) revealed the sequestration of AuNPs in amplified PCR products and the aggregation of AuNPs in non-amplified PCR products, resulting in red and blue colors, respectively. The method was tested on genotyping of six SNPs from six genes (Akr1c3, Plg, Myf5, Sec14l2, Tpm1, and Lama2) in buffaloes, and the results were perfectly matched with those obtained using agarose gel electrophoresis analysis. Therefore, the AS-PCR combined with AuNPs provides an easy visual detection method for the amplified and non-amplified PCR products of single-nucleotide polymorphisms (SNPs). In addition, the presented method has the potential to replace agarose gel electrophoresis, the use of EtBr, and UV-transilluminator.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116142"},"PeriodicalIF":2.5,"publicationDate":"2026-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147832671","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}

引用次数: 0

Recent advances in ZnO-based electrochemical sensors for food safety monitoring 用于食品安全监测的zno电化学传感器研究进展。

IF 2.5 4区生物学

Analytical biochemistry Pub Date : 2026-05-01 Epub Date: 2026-01-08 DOI: 10.1016/j.ab.2026.116049

Md Maruf Ahmed , Qin Xu

{"title":"Recent advances in ZnO-based electrochemical sensors for food safety monitoring","authors":"Md Maruf Ahmed , Qin Xu","doi":"10.1016/j.ab.2026.116049","DOIUrl":"10.1016/j.ab.2026.116049","url":null,"abstract":"<div><div>Food safety is a paramount worldwide issue that directly affects human health and well-being, underscoring the necessity for rapid and accurate detection strategies. Conventional analytical techniques, including chromatography, mass spectrometry, and immunoassays, are highly sensitive and reliable; however, they are costly, laborious, and impractical for real-time monitoring. Conversely, electrochemical sensors have benefits for food safety monitoring, including simplicity, quick response, cost-effectiveness, and portability, making them suitable for continuous monitoring. Optimal sensing performance necessitates the design and development of functional electrode materials with superior electrocatalytic activity. Over the past decade, ZnO nanomaterials have been widely used in electrochemical sensor technology due to their multifunctional properties, including high electron mobility, biocompatibility, and tunable architecture. This review summarizes recent advancements in ZnO-based electrochemical sensors for detecting food toxins. The synthesis methodologies and material characteristics are initially examined, followed by a review of their electrochemical sensing systems. The functionalization strategies for improving ZnO's sensing capabilities have been discussed. This study highlights the existing obstacles and potential opportunities for the advancement of ZnO-based electrochemical sensors in food safety monitoring.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116049"},"PeriodicalIF":2.5,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145948399","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Development and characterization of monoclonal antibodies against inhibin α subunit as a potential cancer biomarker 抑制素α亚基单克隆抗体作为潜在癌症生物标志物的研制和鉴定。

IF 2.5 4区生物学

Analytical biochemistry Pub Date : 2026-05-01 Epub Date: 2026-01-27 DOI: 10.1016/j.ab.2026.116066

Kaoutar Aalilouch , Khalida Sabeur , Ikhlass El Berbri , Faouzi Kichou , Najet Safini , Mehdi Elharrak , Ouafaa Fassi Fihri

{"title":"Development and characterization of monoclonal antibodies against inhibin α subunit as a potential cancer biomarker","authors":"Kaoutar Aalilouch , Khalida Sabeur , Ikhlass El Berbri , Faouzi Kichou , Najet Safini , Mehdi Elharrak , Ouafaa Fassi Fihri","doi":"10.1016/j.ab.2026.116066","DOIUrl":"10.1016/j.ab.2026.116066","url":null,"abstract":"<div><div>Inhibin is a dimeric glycoprotein hormone consisting of α and β subunits. Inhibin has gained significant interest as a biomarker for ovarian and testicular malignancies. While existing inhibin immunoassays have demonstrated efficacy, assays specifically targeting the free α subunit are not yet available. This study aimed to generate monoclonal antibodies against inhibin-α using hybridoma technology, targeting distinct epitopes within the N-region of the α subunit. These epitopes were chosen using bioinformatic analysis to ensure strong antigenicity and successful antibody production. To screen the antibodies, an ELISA was developed using plates coated with recombinant inhibin-α protein. This ELISA confirmed that the antibodies were reactive and could effectively detect inhibin-α <em>in vitro</em>. The antibodies were further evaluated for specificity and immunoreactivity using Western blotting, and IHC methods. Western blot analysis showed that the antibodies specifically recognized the free inhibin-α, demonstrating high specificity under both reducing and non-reducing conditions. IHC studies on normal mouse tissue sections further confirmed the localization of inhibin-α in granulosa, theca, Sertoli, and Leydig cells. The developed antibodies showed significant potential for use <em>in vitro</em> assays for cancer diagnosis. Further validation is needed to ensure their successful clinical application.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116066"},"PeriodicalIF":2.5,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146083778","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}

引用次数: 0

Development of a chemiluminescence immunoassay for proGRP in human serum 人血清中proGRP化学发光免疫分析法的建立。

IF 2.5 4区生物学

Analytical biochemistry Pub Date : 2026-05-01 Epub Date: 2026-02-03 DOI: 10.1016/j.ab.2026.116074

Xiang Chen , Lin Ma , Zhonghu Bai , Jiong Wu

{"title":"Development of a chemiluminescence immunoassay for proGRP in human serum","authors":"Xiang Chen , Lin Ma , Zhonghu Bai , Jiong Wu","doi":"10.1016/j.ab.2026.116074","DOIUrl":"10.1016/j.ab.2026.116074","url":null,"abstract":"<div><div>Progastrin-releasing peptide is a crucial serum biomarker for the diagnosis, differential diagnosis, therapeutic monitoring, and prognostic evaluation of small cell lung cancer. In this study, a double-antibody sandwich immunoassay for the biomarker was developed. Key factors in the method development were systematically investigated, including magnetic particle coating, conjugate labeling, concentrations of magnetic particles and conjugates, and serum sample volume. The established assay has a total analysis time of approximately 13 min, with a limit of blank of 3.53 pg/mL. The repeatability and within-laboratory precision coefficients of variation were less than 8%. Assay accuracy was unaffected by common endogenous interferents at pathological concentrations or by the presence of gastrin-releasing peptide in the serum. Additionally, thermal accelerated stability testing over 7 days confirmed the robust stability of the reagents. Method comparison further demonstrated that the clinical results obtained by the established method showed high consistency with those of the Roche assay, fully meeting the requirements for clinical application.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116074"},"PeriodicalIF":2.5,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146123426","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}

引用次数: 0

Comparing digital and real-time PCR platforms for detecting residual iPSCs and virus-producing cells in manufacturing 比较数字和实时PCR检测制造过程中剩余iPSCs和产病毒细胞的平台。

IF 2.5 4区生物学

Analytical biochemistry Pub Date : 2026-05-01 Epub Date: 2026-01-19 DOI: 10.1016/j.ab.2026.116053

Un Na Koh , Si-Keun Lim

{"title":"Comparing digital and real-time PCR platforms for detecting residual iPSCs and virus-producing cells in manufacturing","authors":"Un Na Koh , Si-Keun Lim","doi":"10.1016/j.ab.2026.116053","DOIUrl":"10.1016/j.ab.2026.116053","url":null,"abstract":"<div><div>Digital PCR (dPCR) enables absolute nucleic acid quantification and has been widely adopted for quality control (QC) applications in cell therapy manufacturing. Ensuring patient safety during cell therapy manufacturing requires reliable detection of trace residual cells, such as undifferentiated induced pluripotent stem cells (iPSCs) and virus-producing cells. This study compared qPCR (CFX96 Opus System, Bio-Rad) with two dPCR platforms—the QX200 Droplet Digital PCR System (Bio-Rad) and the QIAcuity Digital PCR System (QIAGEN). For QC evaluation, iPSCs mixed with differentiated cardiomyocytes (CMs) or neural progenitor cells (NPCs) were analyzed using <em>TDGF1</em>, <em>OCT4</em>, and <em>NANOG</em>, while virus-producing 293T cells in CAR-T preparations were targeted using <em>gag</em> and VSVG sequences within the lentiviral packaging plasmid. Mixed samples were serially diluted from 1:1 to 1:10<sup>6</sup> to evaluate performance across a wide concentration range. Both dPCR platforms and qPCR showed comparable sensitivity and linearity across most dilution points. However, qPCR exhibited more frequent signal loss at low template concentrations. In contrast, dPCR showed reduced variability across dilution intervals, and lower coefficients of variation (CV), indicating more stable quantification at low target levels. Despite minor differences in absolute copy number, both dPCR systems demonstrated comparable analytical performance. These results indicate that, although overall sensitivity and linearity were similar between qPCR and dPCR, dPCR provides more consistent quantification across dilution ranges, supporting its suitability for detecting low-abundance residual cells in cell therapy manufacturing.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116053"},"PeriodicalIF":2.5,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146016843","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}

引用次数: 0

A Word2Vec-ResNet Transfer Learning model for promoter prediction with dimensionality reduction and cross-domain knowledge integration 基于降维和跨领域知识集成的Word2Vec-ResNet启动子预测迁移学习模型。

IF 2.5 4区生物学

Analytical biochemistry Pub Date : 2026-05-01 Epub Date: 2026-02-07 DOI: 10.1016/j.ab.2026.116076

Jiale Fu, Xiao Liu

{"title":"A Word2Vec-ResNet Transfer Learning model for promoter prediction with dimensionality reduction and cross-domain knowledge integration","authors":"Jiale Fu, Xiao Liu","doi":"10.1016/j.ab.2026.116076","DOIUrl":"10.1016/j.ab.2026.116076","url":null,"abstract":"<div><div>Promoter prediction is critical for deciphering transcriptional regulatory mechanisms. However, traditional one-hot encoding strategies suffer from dimensionality explosion with vocabulary expansion, while single-domain knowledge constraints limit predictive performance. Therefore, we propose a promoter prediction method (Word2Vec-ResNet) that innovatively integrates natural language processing (NLP) techniques with cross-domain transfer learning. By pretraining word embeddings on source domain data and transferring the pretrained embedding table to the target domain, the method effectively reduces the dimensionality of nucleotide sequence encoding while leveraging inter-domain knowledge to enhance model generalization. Comprehensive experiments on promoter datasets of four representative organisms (<em>Bacillus subtilis</em>, <em>Escherichia coli</em>, <em>Saccharomyces cerevisiae</em>, and <em>Drosophila melanogaster</em>) demonstrate that the proposed method achieves significant performance improvements: compared with one-hot encoding, its average encoding dimension is reduced by 97.6%; compared with baseline methods, the prediction accuracy is increased by an average of 18.12% (with the ratio of the training set to the test set being 8:2).</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116076"},"PeriodicalIF":2.5,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146140949","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}

引用次数: 0

Engineering GFP–NanoLuc fusion substrates for sensitive, quantitative detection of protease activity via BRET 工程GFP-NanoLuc融合底物，用于通过BRET敏感，定量检测蛋白酶活性

IF 2.5 4区生物学

Analytical biochemistry Pub Date : 2026-05-01 Epub Date: 2026-01-17 DOI: 10.1016/j.ab.2026.116052

Mitsuki Nakamura, Masafumi Sakono

{"title":"Engineering GFP–NanoLuc fusion substrates for sensitive, quantitative detection of protease activity via BRET","authors":"Mitsuki Nakamura, Masafumi Sakono","doi":"10.1016/j.ab.2026.116052","DOIUrl":"10.1016/j.ab.2026.116052","url":null,"abstract":"<div><div>Proteases play essential roles in diverse biological processes and are closely associated with various diseases, making them important targets for diagnostics and therapeutic development. Conventional protease activity assays often rely on chromogenic or fluorogenic substrates that require organic synthesis, increasing production costs and limiting accessibility. To address these, we developed a genetically encoded, bioluminescence resonance energy transfer (BRET)-based protease substrate composed of NanoLuc (NLuc) and green fluorescent protein (GFP) fused to either end of a cleavable peptide sequence. In the intact fusion protein, NLuc luminescence excites GFP via BRET, resulting in green emission. Proteolytic cleavage disrupts BRET, reducing GFP fluorescence. Using tobacco etch virus (TEV) protease as a model, we demonstrated that introducing glycine–serine linkers flanking the cleavage site (TEVcs) enhances proteolytic accessibility and signal responsiveness. The optimized substrate enabled quantitative detection of TEV protease activity with a detection limit of 0.0426 μM. Furthermore, substituting the TEVcs with a caspase-3-specific sequence allowed sensitive detection of caspase-3, with a limit of 0.62 nM. This system offers a cost-effective and broadly applicable platform for real-time protease activity measurement using standard <em>Escherichia coli</em> expression systems, eliminating the need for chemically synthesized substrates.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116052"},"PeriodicalIF":2.5,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145996378","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}

引用次数: 0