Pub Date : 2026-05-01Epub Date: 2026-01-08DOI: 10.1016/j.ab.2026.116049
Md Maruf Ahmed , Qin Xu
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.
{"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}
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 in vitro. 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 in vitro assays for cancer diagnosis. Further validation is needed to ensure their successful clinical application.
{"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}
Pub Date : 2026-05-01Epub Date: 2026-02-03DOI: 10.1016/j.ab.2026.116074
Xiang Chen , Lin Ma , Zhonghu Bai , Jiong Wu
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.
{"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}
Pub Date : 2026-05-01Epub Date: 2026-01-19DOI: 10.1016/j.ab.2026.116053
Un Na Koh , Si-Keun Lim
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 TDGF1, OCT4, and NANOG, while virus-producing 293T cells in CAR-T preparations were targeted using gag and VSVG sequences within the lentiviral packaging plasmid. Mixed samples were serially diluted from 1:1 to 1:106 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.
数字PCR （dPCR）能够实现绝对核酸定量，并已广泛应用于细胞治疗制造中的质量控制（QC）应用。在细胞治疗制造过程中确保患者安全需要可靠地检测微量残留细胞，如未分化的诱导多能干细胞（iPSCs）和产生病毒的细胞。本研究将qPCR （CFX96 Opus System, Bio-Rad）与两种dPCR平台——QX200液滴数字PCR系统（Bio-Rad）和QIAcuity数字PCR系统（QIAGEN）进行了比较。为了进行QC评估，我们使用TDGF1、OCT4和NANOG对混合分化心肌细胞（CMs）或神经祖细胞（npc）的iPSCs进行分析，同时使用慢病毒包装质粒中的gag和VSVG序列对CAR-T制剂中产生病毒的293T细胞进行靶向。混合样品从1:1稀释到1:106，以评估在广泛浓度范围内的性能。dPCR平台和qPCR在大多数稀释点上都显示出相当的灵敏度和线性。然而，在低模板浓度下，qPCR表现出更频繁的信号丢失。相比之下，dPCR在稀释间隔内的变异性较低，变异系数（CV）较低，表明在低目标水平下定量更稳定。尽管绝对拷贝数存在微小差异，但两种dPCR系统均表现出可比较的分析性能。这些结果表明，尽管qPCR和dPCR的总体灵敏度和线性相似，但dPCR在稀释范围内提供了更一致的定量，支持其在细胞治疗制造中检测低丰度残留细胞的适用性。
{"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}
Pub Date : 2026-05-01Epub Date: 2026-02-07DOI: 10.1016/j.ab.2026.116076
Jiale Fu, Xiao Liu
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 (Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster) 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).
{"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}
Pub Date : 2026-05-01Epub Date: 2026-01-17DOI: 10.1016/j.ab.2026.116052
Mitsuki Nakamura, Masafumi Sakono
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 Escherichia coli expression systems, eliminating the need for chemically synthesized substrates.
{"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}
Pub Date : 2026-05-01Epub Date: 2026-02-03DOI: 10.1016/j.ab.2026.116070
Ezgi Salmanli , Taner Karaoglu
Canine parvovirus-2 remains a major threat to canine health, with maternal antibody interference and limitations of traditional diagnostic methods posing challenges to effective disease control. Maternal-derived antibodies can persist at levels that interfere with the development of active immunity following vaccination. Therefore, accurate detection of existing antibody levels prior to vaccination is essential to ensure induction of protective immunity. Hemagglutination inhibition tests are labor-intensive and highly susceptible to external variability, emphasizing the need for reliable and cost-effective alternatives. In this study, we developed and optimized an indirect ELISA using a full-length, recombinant VP2-2b protein expressed in E. coli to detect CPV-2 antibodies. To our knowledge, this is the first ELISA developed around a soluble full-length CPV-2b protein, with comprehensive optimization and clinical validation. VP2-2b was selected for its unique bidirectional neutralization kinetics, enhancing the diagnostic accuracy of CPV-2 serology. The ELISA demonstrated excellent sensitivity and specificity, effectively distinguishing between seropositive and seronegative samples. A gray zone (OD450 = 0.26–0.30) was defined to represent borderline antibody titers, where retesting is recommended to determine accurate vaccination timing. Optimal assay conditions were established as 7.5 μg antigen coating concentration and 1:300 serum dilution, yielding a high signal-to-noise ratio (SNR) of 5.6. Validation against HI tests showed strong correlation with minimal non-specific binding. This ELISA system provides a reliable and economical alternative to traditional methods, helping overcome the limitations of the HI test in field settings and supporting informed decisions in vaccination planning.
{"title":"A recombinant full-length VP2-2b-based ELISA for evaluating immunoprotection against canine parvovirus-2: Expression, development and validation","authors":"Ezgi Salmanli , Taner Karaoglu","doi":"10.1016/j.ab.2026.116070","DOIUrl":"10.1016/j.ab.2026.116070","url":null,"abstract":"<div><div>Canine parvovirus-2 remains a major threat to canine health, with maternal antibody interference and limitations of traditional diagnostic methods posing challenges to effective disease control. Maternal-derived antibodies can persist at levels that interfere with the development of active immunity following vaccination. Therefore, accurate detection of existing antibody levels prior to vaccination is essential to ensure induction of protective immunity. Hemagglutination inhibition tests are labor-intensive and highly susceptible to external variability, emphasizing the need for reliable and cost-effective alternatives. In this study, we developed and optimized an indirect ELISA using a full-length, recombinant VP2-2b protein expressed in <em>E. coli</em> to detect CPV-2 antibodies. To our knowledge, this is the first ELISA developed around a soluble full-length CPV-2b protein, with comprehensive optimization and clinical validation. VP2-2b was selected for its unique bidirectional neutralization kinetics, enhancing the diagnostic accuracy of CPV-2 serology. The ELISA demonstrated excellent sensitivity and specificity, effectively distinguishing between seropositive and seronegative samples. A gray zone (OD450 = 0.26–0.30) was defined to represent borderline antibody titers, where retesting is recommended to determine accurate vaccination timing. Optimal assay conditions were established as 7.5 μg antigen coating concentration and 1:300 serum dilution, yielding a high signal-to-noise ratio (SNR) of 5.6. Validation against HI tests showed strong correlation with minimal non-specific binding. This ELISA system provides a reliable and economical alternative to traditional methods, helping overcome the limitations of the HI test in field settings and supporting informed decisions in vaccination planning.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116070"},"PeriodicalIF":2.5,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146123466","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-05-01Epub Date: 2026-02-06DOI: 10.1016/j.ab.2026.116069
Rui Zhang , Yiyang Tong , Yumeng Shi , Yinuo Cai , Qi Zhao , Yang Lu
Higenamine (HM) is a naturally occurring benzylisoquinoline alkaloid found in plants and is classified as an S3 prohibited substance in the 2020 World Anti-Doping Agency report. To prevent doping violations in competitive sports, it is essential to develop timely and accurate detection methods. In this study, we propose, for the first time, a detection method that combines artificial intelligence (AI)-based image recognition technology with lateral flow immunoassay (LFIA). This method utilizes gold nanoparticles (AuNPs) conjugated with HM-specific antibodies for the rapid detection of HM in urine via LFIA. It integrates AI-based image recognition to quantitatively analyze color intensity of the test strip's detection line. Experimental results demonstrated that the proposed method achieves a limit of detection of 0.49 ng/mL, enabling accurate identification within the concentration range of 2–8 ng/mL. For model training, an Image Classification of HM Test strip dataset was created. To address the limitations of this dataset, namely its small size (n = 304) and limited feature diversity, an advanced solution tailored to these constraints was proposed. The use of Contrast Limited Adaptive Histogram Equalization (CLAHE), which enables finer-grained feature extraction, ensured excellent recognition accuracy of the model in this dataset. During actual urine detection, the model achieved a prediction accuracy of 96.88% on the test set. This approach provides an efficient and reliable technical solution for on-site rapid detection of HM, addressing the critical need for timely monitoring in anti-doping efforts.
{"title":"Development of an AI image Recognition–based lateral flow immunochromatographic test strip for higenamine detection","authors":"Rui Zhang , Yiyang Tong , Yumeng Shi , Yinuo Cai , Qi Zhao , Yang Lu","doi":"10.1016/j.ab.2026.116069","DOIUrl":"10.1016/j.ab.2026.116069","url":null,"abstract":"<div><div>Higenamine (HM) is a naturally occurring benzylisoquinoline alkaloid found in plants and is classified as an S3 prohibited substance in the 2020 World Anti-Doping Agency report. To prevent doping violations in competitive sports, it is essential to develop timely and accurate detection methods. In this study, we propose, for the first time, a detection method that combines artificial intelligence (AI)-based image recognition technology with lateral flow immunoassay (LFIA). This method utilizes gold nanoparticles (AuNPs) conjugated with HM-specific antibodies for the rapid detection of HM in urine via LFIA. It integrates AI-based image recognition to quantitatively analyze color intensity of the test strip's detection line. Experimental results demonstrated that the proposed method achieves a limit of detection of 0.49 ng/mL, enabling accurate identification within the concentration range of 2–8 ng/mL. For model training, an Image Classification of HM Test strip dataset was created. To address the limitations of this dataset, namely its small size (n = 304) and limited feature diversity, an advanced solution tailored to these constraints was proposed. The use of Contrast Limited Adaptive Histogram Equalization (CLAHE), which enables finer-grained feature extraction, ensured excellent recognition accuracy of the model in this dataset. During actual urine detection, the model achieved a prediction accuracy of 96.88% on the test set. This approach provides an efficient and reliable technical solution for on-site rapid detection of HM, addressing the critical need for timely monitoring in anti-doping efforts.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116069"},"PeriodicalIF":2.5,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146140947","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-05-01Epub Date: 2026-01-30DOI: 10.1016/j.ab.2026.116064
Jessa D. Alonzo , Kristopher Ray S. Pamintuan , Khyle Glainmer N. Quiton , Jyh Jian Chen
Acute hepatopancreatic necrosis disease (AHPND), caused by Vibrio parahaemolyticus (Vp) carrying pirvpA/pirvpB, continues to threaten shrimp aquaculture across Asia. This study demonstrates a laboratory proof-of-concept gold nanoparticle-based (AuNPs) colorimetric biosensor targeting the pirvpA gene of Vp. Citrate-reduced AuNPs (∼15.54 nm; SPR ≈ 520 nm) were functionalized with thiolated ssDNA probes (AuNP-Probe) using a deoxyadenosine triphosphate-assisted immobilization strategy and subsequently stabilized with low molecular weight (LMW) chitosan (AuNP-Probe-Chit). Loop-mediated isothermal amplification (LAMP) generated the target DNA, while detection relied on salt-induced aggregation assessed by visual color/precipitate formation and UV–Vis spectral shifts (A650/Aλmax, λmax ≈ 532 nm for AuNP-Probe-Chit). Optimization identified 1.0 μM probe and 0.01% (v/v) chitosan as the optimal conditions for forming stable conjugates. Upon hybridization, an 8:2 (v/v) AuNP-Probe-Chit:LAMP mixture, combined with 0.5 M NaCl, enabled clear discrimination between positive and negative samples. Positives retained a pink-red supernatant with minimal aggregation, whereas negatives formed precipitates and exhibited higher aggregation indices. The biosensor demonstrated strong specificity for pirvpA-positive DNA, without cross-reacting with Vibrio vulnificus or Vibrio alginolyticus, and exhibited sensitivity approaching the LAMP amplification threshold, with reliable discrimination observed at ≥10−1 ng of target DNA. These findings establish a robust, equipment-lean diagnostic platform that integrates sequence-specific hybridization with polymer-mediated colloidal stabilization. The AuNP-Probe-Chit system offers rapid, visible discrimination, highlighting its potential for future AHPND surveillance in resource-limited settings. Compared with previous works, the incorporation of thiolated ssDNA probe functionalization with chitosan stabilization within a single AuNPs-based platform tailored for aquaculture pathogens, such as Vp, is currently underexplored, highlighting a significant need that this study aims to address.
{"title":"Development of LMW chitosan-stabilized gold nanoparticle DNA biosensors functionalized with thiolated probe for Vibrio parahaemolyticus detection","authors":"Jessa D. Alonzo , Kristopher Ray S. Pamintuan , Khyle Glainmer N. Quiton , Jyh Jian Chen","doi":"10.1016/j.ab.2026.116064","DOIUrl":"10.1016/j.ab.2026.116064","url":null,"abstract":"<div><div>Acute hepatopancreatic necrosis disease (AHPND), caused by <em>Vibrio parahaemolyticus</em> (Vp) carrying <em>pir</em><sup><em>vp</em></sup><em>A/pir</em><sup><em>vp</em></sup><em>B</em>, continues to threaten shrimp aquaculture across Asia. This study demonstrates a laboratory proof-of-concept gold nanoparticle-based (AuNPs) colorimetric biosensor targeting the <em>pir</em><sup><em>vp</em></sup><em>A</em> gene of Vp. Citrate-reduced AuNPs (∼15.54 nm; SPR ≈ 520 nm) were functionalized with thiolated ssDNA probes (AuNP-Probe) using a deoxyadenosine triphosphate-assisted immobilization strategy and subsequently stabilized with low molecular weight (LMW) chitosan (AuNP-Probe-Chit). Loop-mediated isothermal amplification (LAMP) generated the target DNA, while detection relied on salt-induced aggregation assessed by visual color/precipitate formation and UV–Vis spectral shifts (A<sub>65</sub>0/A<sub>λmax</sub>, <em>λ</em><sub>max</sub> ≈ 532 nm for AuNP-Probe-Chit). Optimization identified 1.0 μM probe and 0.01% (v/v) chitosan as the optimal conditions for forming stable conjugates. Upon hybridization, an 8:2 (v/v) AuNP-Probe-Chit:LAMP mixture, combined with 0.5 M NaCl, enabled clear discrimination between positive and negative samples. Positives retained a pink-red supernatant with minimal aggregation, whereas negatives formed precipitates and exhibited higher aggregation indices. The biosensor demonstrated strong specificity for <em>pir</em><sup><em>vp</em></sup><em>A</em>-positive DNA, without cross-reacting with <em>Vibrio vulnificus</em> or <em>Vibrio alginolyticus</em>, and exhibited sensitivity approaching the LAMP amplification threshold, with reliable discrimination observed at ≥10<sup>−1</sup> ng of target DNA. These findings establish a robust, equipment-lean diagnostic platform that integrates sequence-specific hybridization with polymer-mediated colloidal stabilization. The AuNP-Probe-Chit system offers rapid, visible discrimination, highlighting its potential for future AHPND surveillance in resource-limited settings. Compared with previous works, the incorporation of thiolated ssDNA probe functionalization with chitosan stabilization within a single AuNPs-based platform tailored for aquaculture pathogens, such as Vp, is currently underexplored, highlighting a significant need that this study aims to address.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116064"},"PeriodicalIF":2.5,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146099535","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}
Helicobacter pylori, a Group I carcinogen that infects over 50% of the global population, is a major contributor to gastric cancer and the growing crisis of antibiotic resistance. However, vaccine development has remained challenging due to the poor immunogenicity of candidate antigens and limitations in delivery strategies. To address this, we engineered a novel Fc-fusion vaccine targeting antigen-presenting cells (APCs) by fusing H. pylori UreB317-329 and UreB409-421 epitopes with Lpp20 lipoprotein to the murine IgG2a Fc domain (UreB: Lpp20: mFcγ2a). This study presents a preliminary, proof-of-concept evaluation of an Fc-fusion epitope-based vaccine candidate, focusing on in vitro characterization and APC-targeting properties. The construct was codon-optimized, expressed in Pichia pastoris, and purified via affinity chromatography. Expression levels in the mg/mL range, as determined by biochemical analysis, with >95% purity confirmed by SDS-PAGE and Western blot. Co-localization assays demonstrated 4.2-fold higher APC uptake of the Fc-fusion protein versus a His-tagged control (p < 0.001, unpaired t-test), mediated by FcγRI (CD64) binding. The protein maintained its structural stability for 30 days at 4 °C (<5% batch variability), underscoring manufacturing feasibility. While in vitro results validate APC targeting, limitations include preclinical scope and murine Fc compatibility gaps for human translation. This study established P. pastoris as a scalable platform for Fc-fusion vaccines, bypassing adjuvants and enhancing immunogenicity through conserved epitopes. Future work must evaluate in vivo efficacy in infection models and human Fc adaptations. These findings offer a blueprint for next-generation vaccines against H. pylori and other intracellular pathogens, urgently needed in antimicrobial resistance containment strategies.
{"title":"Design and in vitro evaluation of an Fc-fusion epitope-based vaccine candidate against Helicobacter pylori","authors":"Roghayeh Mohammadzadeh , Shaho Menbari , Janbibi Dorazehi , Mojtaba Sankian , Hadi Farsiani","doi":"10.1016/j.ab.2026.116071","DOIUrl":"10.1016/j.ab.2026.116071","url":null,"abstract":"<div><div><em>Helicobacter pylori</em>, a Group I carcinogen that infects over 50% of the global population, is a major contributor to gastric cancer and the growing crisis of antibiotic resistance. However, vaccine development has remained challenging due to the poor immunogenicity of candidate antigens and limitations in delivery strategies. To address this, we engineered a novel Fc-fusion vaccine targeting antigen-presenting cells (APCs) by fusing <em>H. pylori</em> UreB<sub>317</sub>-<sub>329</sub> and UreB<sub>409</sub>-<sub>421</sub> epitopes with Lpp20 lipoprotein to the murine IgG2a Fc domain (UreB: Lpp20: mFcγ2a). This study presents a preliminary, proof-of-concept evaluation of an Fc-fusion epitope-based vaccine candidate, focusing on <em>in vitro</em> characterization and APC-targeting properties. The construct was codon-optimized, expressed in <em>Pichia pastoris</em>, and purified via affinity chromatography. Expression levels in the mg/mL range, as determined by biochemical analysis, with >95% purity confirmed by SDS-PAGE and Western blot. Co-localization assays demonstrated 4.2-fold higher APC uptake of the Fc-fusion protein versus a His-tagged control (<em>p</em> < 0.001, unpaired <em>t</em>-test), mediated by FcγRI (CD64) binding. The protein maintained its structural stability for 30 days at 4 °C (<5% batch variability), underscoring manufacturing feasibility. While <em>in vitro</em> results validate APC targeting, limitations include preclinical scope and murine Fc compatibility gaps for human translation. This study established <em>P. pastoris</em> as a scalable platform for Fc-fusion vaccines, bypassing adjuvants and enhancing immunogenicity through conserved epitopes. Future work must evaluate <em>in vivo</em> efficacy in infection models and human Fc adaptations. These findings offer a blueprint for next-generation vaccines against <em>H. pylori</em> and other intracellular pathogens, urgently needed in antimicrobial resistance containment strategies.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116071"},"PeriodicalIF":2.5,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146117643","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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