Analytical biochemistry最新文献

Expression of concern: "Specific detection of Mycobacterium sp. genomic DNA using dual labeled gold nanoparticle based electrochemical biosensor" [Anal. Biochem. 417 (1) (2011) 73-79]. 关注表达：“使用基于双标记金纳米粒子的电化学生物传感器特异性检测分枝杆菌基因组DNA”[肛。生物化学，2011(1):73-79。

IF 3.1 4区生物学

Analytical biochemistry Pub Date : 2026-10-01 Epub Date: 2026-06-25 DOI: 10.1016/j.ab.2026.116186

引用次数: 0

A CRISPR/Cas9 multiplex nucleic acid sensing platform based on rolling circle amplification-mediated monomeric substrate generation. 基于滚动环扩增介导的单体底物生成的CRISPR/Cas9多重核酸传感平台

IF 3.1 4区生物学

Analytical biochemistry Pub Date : 2026-09-03 DOI: 10.1016/j.ab.2026.116249

Wei Li

{"title":"A CRISPR/Cas9 multiplex nucleic acid sensing platform based on rolling circle amplification-mediated monomeric substrate generation.","authors":"Wei Li","doi":"10.1016/j.ab.2026.116249","DOIUrl":"10.1016/j.ab.2026.116249","url":null,"abstract":"<p><p>The multiplex detection of nucleic acid biomarkers, including circulating tumor DNAs (ctDNAs) and microRNAs (miRNAs), holds promise for improving the accuracy of molecular diagnostic techniques. In this study, we developed a CRISPR/Cas9-based sensing platform capable of simultaneously detecting multiple nucleic acid biomarkers. This approach leverages the activation of the CRISPR/Cas9 system through the generation of monomeric substrates mediated by rolling circle amplification (RCA). Specifically, upon the presence of a specific target, the RCA process is initiated, producing concatemeric DNA products. By incorporating an endonuclease and complementary reporter molecules, the RCA products are cleaved into monomeric substrates. Subsequently, Cas9/sgRNA complexes recognize and bind these monomeric substrates, activating the CRISPR/Cas9 systems, which further cleave the substrates to produce amplified fluorescence signals. Compared to conventional RCA methods, which often suffer from product aggregation, the monomeric substrate generation strategy demonstrated enhanced sensing capabilities and analytical performance, with signal intensities nearly double those observed in traditional approaches. To enable multiplex detection, three distinct sets of padlock probes, reporter molecules, and sgRNAs were designed to selectively target three different nucleic acid sequences. The platform exhibited high sensitivity and simultaneous detection of multiple ctDNAs and miRNAs, achieving limits of detection (LODs) ranging from 1.28 fM to 1.50 fM for ctDNAs and 0.61 fM to 0.97 fM for miRNAs. Additionally, analyses of clinical serum samples confirmed the method's accuracy and applicability within complex biological matrices. Collectively, this work introduces a multiplex nucleic acid biomarker detection platform with significant potential for advancing molecular sensing and clinical diagnostic applications.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116249"},"PeriodicalIF":3.1,"publicationDate":"2026-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148885986","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

Selective fluorescence turn-on detection of methylglyoxal using N-substituted NBD derivatives. n-取代NBD衍生物选择性荧光开启检测甲基乙二醛。

IF 3.1 4区生物学

Analytical biochemistry Pub Date : 2026-09-01 DOI: 10.1016/j.ab.2026.116242

Adrien Moragues, Léo Jingeot, Camille Nguyen, Cédric Spitz, David Bergé-Lefranc, Miquel Huix-Rotllant, Sylvia Pietri, Pierre Stocker

{"title":"Selective fluorescence turn-on detection of methylglyoxal using N-substituted NBD derivatives.","authors":"Adrien Moragues, Léo Jingeot, Camille Nguyen, Cédric Spitz, David Bergé-Lefranc, Miquel Huix-Rotllant, Sylvia Pietri, Pierre Stocker","doi":"10.1016/j.ab.2026.116242","DOIUrl":"10.1016/j.ab.2026.116242","url":null,"abstract":"<p><p>Methylglyoxal (MGO) is a reactive endogenous α-dicarbonyl involved in glycation, oxidative stress, and metabolic disease. Its measurement in biological samples remains challenging because conventional assays generally require derivatization, chromatographic separation, and costly instrumentation. Fluorescence detection offers a simpler alternative, but currently available probes often lack selectivity for MGO over other biologically relevant carbonyl compounds. We therefore developed a series of N-substituted 4-hydrazino-7-nitrobenzofurazan derivatives for fluorimetric detection of MGO. Alkyl groups were introduced on the hydrazine nitrogen to modulate probe reactivity, steric accessibility, and the fluorescent properties of the resulting hydrazones. The probes were characterized by fluorescence spectroscopy, HPLC with fluorescence detection, LC-MS, and density functional theory calculations. Compared with unsubstituted NBD-H, most N-substituted derivatives produced stronger and more stable responses to MGO, while showing weaker responses to competing carbonyl compounds. Chromatographic and mass spectrometric analyses confirmed formation of the expected probe-MGO hydrazones, and calculations supported preferential condensation at the aldehyde group of MGO. In human plasma, most N-substituted probes retained a strong and highly linear fluorescence response to MGO, even in the presence of biologically relevant carbonyl interferents such as glyoxal, acrolein, acetaldehyde, and formaldehyde. Compared with unsubstituted NBD-H, these derivatives markedly improved analytical sensitivity toward MGO, supporting their use for fluorimetric detection in complex biological matrices. These findings support a microplate-compatible fluorimetric assay for MGO requiring only a protein-precipitation step and avoiding chromatographic separation or extensive sample clean-up.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116242"},"PeriodicalIF":3.1,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148872713","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

Identification of MDV and REV Using RPA-CRISPR/Cas12a Combined with Fluorescence and Lateral Flow Assays. 利用RPA-CRISPR/Cas12a结合荧光和侧流法鉴定MDV和REV。

IF 3.1 4区生物学

Analytical biochemistry Pub Date : 2026-09-01 DOI: 10.1016/j.ab.2026.116240

Ruiying Han, Ning Xiao, Jiahua Wang, Zhirui Wu, Xiaoli Wang, Xiaochuan Tang

{"title":"Identification of MDV and REV Using RPA-CRISPR/Cas12a Combined with Fluorescence and Lateral Flow Assays.","authors":"Ruiying Han, Ning Xiao, Jiahua Wang, Zhirui Wu, Xiaoli Wang, Xiaochuan Tang","doi":"10.1016/j.ab.2026.116240","DOIUrl":"https://doi.org/10.1016/j.ab.2026.116240","url":null,"abstract":"<p><p>Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) are two major immunosuppressive oncogenic viruses that severely threaten the global poultry industry. Field co-infection of these two pathogens is prevalent, accompanied by highly similar pathological lesions, making conventional diagnostic technologies incapable of rapid on-site differential identification. Recombinase polymerase amplification (RPA) coupled with the CRISPR/Cas12a system represents an innovative platform for rapid nucleic acid detection. In this study, target-specific crRNAs were designed against the conserved unique genes of MDV and REV, respectively, to establish a CRISPR-based diagnostic system capable of differentiating the two viruses for the first time. Comprehensive optimization of RPA reaction composition, buffer formulation and incubation temperature improved the overall detection performance. Three independent visual signal readout modalities were supported by this assay, including real-time fluorescence quantification, naked-eye fluorescence visualization under blue light, and lateral flow assay (LFA). Sensitivity evaluation demonstrated that the limit of detection (LOD) reached 1×10<sup>0</sup> copies/μL for MDV and 1×10<sup>1</sup> copies/μL for REV. Specificity assays verified that the system only generated specific positive signals in response to target viruses without cross-reactivity against other common avian oncogenic viruses and their subgroups. Blind testing of 30 clinical chick fecal specimens revealed that the assay achieved a 100% positive detection rate for both viruses when referenced to qPCR and RT-qPCR, with outstanding diagnostic efficacy and perfect detection consistency. Collectively, the developed RPA-CRISPR/Cas12a assay overcomes the limitations of traditional diagnostic methods and holds great promise for on-site routine screening in poultry farms and epidemiological surveillance of MDV and REV.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116240"},"PeriodicalIF":3.1,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148872667","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

Simultaneous GC-MS determination of unsubstituted, hydroxy-, amino-, and hydroxy-amino short-chain fatty acids together with amino acids in biological and food samples following derivatization. 同时气相色谱-质谱法测定生物和食品样品衍生后的非取代、羟基、氨基和羟基氨基短链脂肪酸及氨基酸。

IF 3.1 4区生物学

Analytical biochemistry Pub Date : 2026-08-31 DOI: 10.1016/j.ab.2026.116241

Kalliroi Maria Katsari, Constantine D Stalikas

{"title":"Simultaneous GC-MS determination of unsubstituted, hydroxy-, amino-, and hydroxy-amino short-chain fatty acids together with amino acids in biological and food samples following derivatization.","authors":"Kalliroi Maria Katsari, Constantine D Stalikas","doi":"10.1016/j.ab.2026.116241","DOIUrl":"10.1016/j.ab.2026.116241","url":null,"abstract":"<p><p>The simultaneous determination of short-chain fatty acids (SCFAs) and amino acids is of increasing interest, as these metabolite classes are important indicators of host metabolism and gut microbiota activity. They also serve as biomarkers for disease diagnosis and indicators of food composition. In this study, a gas chromatography-mass spectrometry method was developed for the simultaneous determination of both substituted (hydroxy-, amino- and hydroxy-amino-) and unsubstituted SCFAs, alongside amino acids, following derivatization and liquid-liquid extraction. Isobutyl chloroformate was employed as the derivatization reagent. The proposed method demonstrated excellent linearity, with coefficients of determination (R<sup>2</sup>) ranging from 0.9840 to 0.9992. Method limits of detection ranged from 0.011 to 7.2 μg/mL, while method limits of quantification ranged from 0.033 to 8.0 μg/mL. Intra-day precision (%RSD) ranged from 1.0% to 6.5% for biological samples and from 1.1% to 4.5% for eggs. Inter-day precision (%RSD) ranged from 1.0% to 5.6% for biological samples and from 2.0% to 6.7% for eggs. Matrix effects ranged from 98% to 108% for biological samples and from 96% to 113% for eggs, while recoveries ranged from 90% to 111% and from 95% to 108%, respectively. To the best of our knowledge, this is the first GC-MS method enabling the simultaneous determination of substituted and unsubstituted SCFAs together with amino acids in a single analytical procedure. The proposed approach provides a reliable and versatile platform for metabolomics, microbiome research, clinical investigations, and food analysis, while also providing considerable potential for expansion to a wider range of target metabolites.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116241"},"PeriodicalIF":3.1,"publicationDate":"2026-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148863232","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

Validation of SARS-CoV-2 neutralization assay using VSV-based pseudovirus system. 基于vsv假病毒系统的SARS-CoV-2中和试验验证。

IF 3.1 4区生物学

Analytical biochemistry Pub Date : 2026-08-30 DOI: 10.1016/j.ab.2026.116239

Anita Artarini, Marselina Irasonia Tan, Ernawati Arifin Giri-Rachman, Dessy Natalia, David Virya Chen, Chikako Ono, Yoshiharu Matsuura

{"title":"Validation of SARS-CoV-2 neutralization assay using VSV-based pseudovirus system.","authors":"Anita Artarini, Marselina Irasonia Tan, Ernawati Arifin Giri-Rachman, Dessy Natalia, David Virya Chen, Chikako Ono, Yoshiharu Matsuura","doi":"10.1016/j.ab.2026.116239","DOIUrl":"10.1016/j.ab.2026.116239","url":null,"abstract":"<p><p>The gold standard for SARS-CoV-2 neutralization assays involves wild-type virus, which requires Biosafety Level 3 (BSL-3) containment. To improve safety and accessibility, pseudovirus-based neutralization assays utilizing non-replicating particles like Vesicular Stomatitis Virus (VSV) expressing the SARS-CoV-2 spike protein can be conducted under BSL-2 conditions. This study aimed to perform the analytical validation of a VSV-based pseudovirus system for SARS-CoV-2 using recombinant monoclonal antibody. Pseudo-VSV carrying SARS-CoV-2 Spike proteins were produced using LentiX-293T cells. The assay system was optimized for Multiplicity of Infection (MOI) and assessed for specificity, limit of quantification (LOQ), linearity, accuracy, and precision using the neutralizing mAb BD-604. The system was optimized at an MOI of 0.25. The assay proved highly specific, as mAb BD-604 showed clear neutralizing activity while mAb 1A9 did not. The limit of quantification (LOQ) was determined to be 125 ng of mAb BD-604, with a linear range of 125 - 1000 ng. The relative accuracy remained within the 80-120% range, and the precision (%CV) ranged from 1.92% to 13.57%. Additionally, the system successfully characterized variant-specific neutralization, revealing that BD-604 was effective against the Wuhan, Delta, and Omicron BA.1/BA.2 strains but lacked activity against the Omicron XBB.1.5 variant. This validated pseudo-VSV based SARS-CoV-2 neutralization assay is a valuable bioassay for evaluating neutralizing antibody potency against various SARS-CoV-2 strains in a BSL-2 environment, thus making it useful for vaccine and therapeutic development.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116239"},"PeriodicalIF":3.1,"publicationDate":"2026-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148860392","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 validation of a mechanism-based Ba/F3-hGHR bioassay for the potency assessment of recombinant human growth hormone. 基于Ba/F3-hGHR机制的重组人生长激素效价测定方法的建立与验证。

IF 3.1 4区生物学

Analytical biochemistry Pub Date : 2026-08-27 DOI: 10.1016/j.ab.2026.116237

Jun Feng, Tao Luo, Chen Guo, Xue Feng, Jianguang Lu, Yuanzhen Dong

{"title":"Development and validation of a mechanism-based Ba/F3-hGHR bioassay for the potency assessment of recombinant human growth hormone.","authors":"Jun Feng, Tao Luo, Chen Guo, Xue Feng, Jianguang Lu, Yuanzhen Dong","doi":"10.1016/j.ab.2026.116237","DOIUrl":"10.1016/j.ab.2026.116237","url":null,"abstract":"<p><p>The biological potency of recombinant human growth hormone (rhGH) is a critical quality attribute (CQA) for ensuring clinical efficacy. However, current pharmacopoeial methods, such as the USP in vitro Nb2-11 cell proliferation assay, lack specificity as they measure lactogenic activity via prolactin receptors rather than the growth hormone receptor (GHR). In this study, we developed a highly specific and robust potency assay for rhGH by standardizing and validating a murine pro-B lymphocyte (Ba/F3) cell platform stably expressing human GHR (hGHR). While GHR-transfected cell models have been previously described for basic research, this work bridges the gap between research-grade tools and pharmaceutical quality control (QC). We established a strictly characterized monoclonal cell line to ensure the long-term genetic and functional stability essential for routine commercial testing. The engineered Ba/F3-hGHR cells exhibited a dose-dependent proliferative response exclusively triggered by hGHR signaling. Through systematic optimization of assay parameters-including cell seeding density, serum concentrations, and incubation kinetics-we established a high-performance analytical protocol. The method was rigorously validated according to ICH Q2(R2) guidelines, demonstrating superior intermediate precision (GCV 8.4-18.3%), high accuracy, and the sensitivity required to detect subtle potency shifts in degraded samples. Our findings provide a mechanism-based, validated alternative to current regulatory methods, offering a reliable approach for the accurate quantification of rhGH biological activity in biopharmaceutical manufacturing.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116237"},"PeriodicalIF":3.1,"publicationDate":"2026-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148839188","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

Camel milk-derived extracellular vesicles using a two-step PEG precipitation strategy. 使用两步聚乙二醇沉淀策略的骆驼乳衍生的细胞外囊泡。

IF 3.1 4区生物学

Analytical biochemistry Pub Date : 2026-08-26 DOI: 10.1016/j.ab.2026.116235

Hanieh Goudarzian, Yahya Sefidbakht, Vuk Uskoković

{"title":"Camel milk-derived extracellular vesicles using a two-step PEG precipitation strategy.","authors":"Hanieh Goudarzian, Yahya Sefidbakht, Vuk Uskoković","doi":"10.1016/j.ab.2026.116235","DOIUrl":"10.1016/j.ab.2026.116235","url":null,"abstract":"<p><p>Isolation of extracellular vesicles (EVs) from milk is technically challenging due to the presence of high concentrations of casein and other proteinaceous contaminants. In this study, we report an optimized two-step polyethylene glycol (PEG) precipitation protocol designed to improve the recovery and enrichment of camel milk-derived EVs. The performance of this two-step protocol, in which initial precipitation at PEG10% is followed by re-precipitation at Re-PEG5%, was evaluated and compared against several alternative isolation strategies, including single-and two-step PEG10% precipitation, and PEG precipitation combined with ultrafiltration (UF) and size-exclusion chromatography (SEC), with protocols assessed across multiple parameters. A suite of quantitative assessments, including protein-to-lipid and nucleic acid-to-protein ratio measurements and flow cytometry, was performed alongside qualitative characterizations via TEM, SEM, AFM, ATR-FTIR, NMR, DSC, EDS and elemental mapping. Complementary characterizations consistently demonstrated that the two-step Re-PEG5% protocol generated EV preparations with higher purity and reduced co-isolation of non-vesicular contaminants than the two-step PEG10% protocol or protocols involving costlier UF and SEC techniques. This improved purity was accompanied by enhanced cytocompatibility in HEK293 cells, as verified through MTT assays, suggesting that optimization of PEG concentration is a critical determinant of both camel milk EV quality and biological performance. Overall, the physicochemical and biological characterizations performed provide substantial evidence supporting the quality and yield of the EVs isolated from camel milk using a two-step PEG precipitation protocol. This cost-effective and scalable strategy offers a promising, green-chemistry-aligned alternative for EV production in biomedical applications, particularly in resource-limited settings.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116235"},"PeriodicalIF":3.1,"publicationDate":"2026-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148823980","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

An indole-oxadiazole-derived fluorescent probe for the detection of the human serum albumin level in thoracic surgery 一种用于胸外科手术中检测人血清白蛋白水平的吲哚-恶二唑衍生荧光探针。

IF 2.5 4区生物学

Analytical biochemistry Pub Date : 2026-06-01 Epub Date: 2026-02-22 DOI: 10.1016/j.ab.2026.116090

Hanzhang Sun , Guanglei Zuo , Yueming Wu , Yangyong Lou , Jiang Feng , Yulong Zheng

{"title":"An indole-oxadiazole-derived fluorescent probe for the detection of the human serum albumin level in thoracic surgery","authors":"Hanzhang Sun , Guanglei Zuo , Yueming Wu , Yangyong Lou , Jiang Feng , Yulong Zheng","doi":"10.1016/j.ab.2026.116090","DOIUrl":"10.1016/j.ab.2026.116090","url":null,"abstract":"<div><div>In this work, the fluorescent probe <strong>InOx-HSA</strong> was developed from an indole-oxadiazole derivative for the detection of the HSA level in thoracic surgery. The probe reported a ratio fluorescence response from 405 nm to 505 nm under the excitation at 310 nm. <strong>InOx-HSA</strong> occupied the binding site of phenylbutazone in HSA and generated the fluorescence enhancement via Förster resonance energy transfer/twisted intramolecular charge transfer mechanisms. The response between <strong>InOx-HSA</strong> and HSA completed within 30 min. The probe-HSA complex kept stable within the range of 7.0-9.0. The ratio response was in a dosage-dependent manner with the limit of detection as 0.0002 mg/mL. <strong>InOx-HSA</strong> exhibited high specificity and low cytotoxicity. The site-binding strategy was confirmed by the drug competition, trypsin digestion, and protein denaturation by high temperature. <strong>InOx-HSA</strong> achieved visualizing HSA in living pulmonary cells, and revealed the ventilation-related consumption of HSA during the thoracic surgery.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"713 ","pages":"Article 116090"},"PeriodicalIF":2.5,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147282090","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

Imidazole stabilizes NAD(P)H and improves reductase assays under challenging conditions 咪唑稳定NAD(P)H和改善还原酶在挑战性条件下的测定。

IF 2.5 4区生物学

Analytical biochemistry Pub Date : 2026-06-01 Epub Date: 2026-02-28 DOI: 10.1016/j.ab.2026.116094

Shengkai Yin , Litao Wang , Xian'ai Shi , Guozeng Wang

{"title":"Imidazole stabilizes NAD(P)H and improves reductase assays under challenging conditions","authors":"Shengkai Yin , Litao Wang , Xian'ai Shi , Guozeng Wang","doi":"10.1016/j.ab.2026.116094","DOIUrl":"10.1016/j.ab.2026.116094","url":null,"abstract":"<div><div>The reduced form of nicotinamide adenine dinucleotide (NAD(P)H) is prone to hydrolysis and ring-opening degradation under high-temperature and acidic conditions, resulting in a loss of reducing power and substantial inaccuracies in enzymatic assays. In this study, we systematically investigated the stabilizing effect of imidazole on NADH and NADPH under elevated temperature (30–60 °C) and acidic (pH 3–5) conditions, and further evaluated its applicability in reductase activity assays. Incubation experiments with varying imidazole concentrations (0–300 mM) demonstrated a pronounced concentration-dependent stabilization of NAD(P)H. Enzymatic assays revealed that imidazole enhanced the apparent activities of lactate dehydrogenase (LDH) and imine reductase (IRED) under both acidic and high-temperature conditions. Moreover, imidazole addition broadened the optimal pH and temperature range of these enzymes, markedly improving the accuracy and reproducibility of activity measurements. Collectively, these findings identify imidazole as an efficient stabilizing additive for NAD(P)H, enabling more reliable assessment of NAD(P)H-dependent reductase activities under high-temperature or acidic conditions.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"713 ","pages":"Article 116094"},"PeriodicalIF":2.5,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147324467","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