Analytical biochemistry最新文献_第6页

Validation of assay for measuring acetyl-coenzyme a carboxylase activity in grasses using malachite green 利用孔雀石绿验证测定草中乙酰辅酶 A 羧化酶活性的方法

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-11-23 DOI: 10.1016/j.ab.2024.115723

Yoshinobu Jin

Acetyl-CoA carboxylase (ACCase) is one of the most important enzymes as a herbicide target in gramineous plant species, however, assay methods for the enzyme are primarily limited to those using radioisotopes (RI). Typically, the measurement method that uses RI necessitates specialized facilities and equipment, and involves complex procedures throughout the experiment. As another method for detecting ACCase activity, the colorimetric method using malachite green (MG) is also known. However, reports on this method are limited, and information regarding the simplicity of the procedure and the scope of its application remains unclear. To better understand the method using MG and to develop a simpler assay method, crude enzymes extracted from various target-site resistant (TSR) biotypes of blackgrass (Alopecurus myosuroides) were examined in enzyme inhibition tests. As a result, this method was able to accurately detect the relationship between the chemical classes of ACC inhibitors and cross-resistance to specific TSRs. Moreover, the ACCase activity of other grass species was also examined using this method. By using crude enzymes from various species and a commercially available phosphatase kit containing MG, ACCase activity was detectable with good accuracy. In addition, enzyme inhibition studies using ACCase inhibiting herbicides revealed that this method reproduced results similar to those obtained with the RI method. The Z′-factor, an index of high-throughput screening (HTS), was around 0.7, indicating that it is an excellent screening system. These results suggest that the assay method using MG is very simple, labor-saving, and accurate with a throughput much higher than that of the existing RI method. Therefore, it is strongly suggested that the method could replace the RI method in most cases. These results indicate that it is applicable to HTS for ACCase inhibitors.

乙酰-CoA羧化酶（ACCase）是禾本科植物中作为除草剂靶标的最重要的酶之一，但该酶的检测方法主要局限于使用放射性同位素（RI）的方法。通常，使用放射性同位素的测量方法需要专门的设施和设备，而且整个实验过程涉及复杂的程序。另一种检测 ACCase 活性的方法是使用孔雀石绿（MG）的比色法。然而，有关这种方法的报道并不多，而且有关该方法的简易性及其应用范围的信息仍不明确。为了更好地理解使用孔雀石绿的方法，并开发出一种更简单的检测方法，我们在酶抑制试验中检测了从黑草（Alopecurus myosuroides）的各种抗靶点（TSR）生物型中提取的粗酶。结果，这种方法能够准确检测 ACC 抑制剂化学类别与对特定 TSR 的交叉抗性之间的关系。此外，该方法还检测了其他草种的 ACC 酶活性。通过使用来自不同草种的粗酶和含有 MG 的市售磷酸酶试剂盒，可以很准确地检测到 ACC 酶活性。此外，使用抑制 ACCase 的除草剂进行的酶抑制研究表明，该方法可再现与 RI 方法类似的结果。作为高通量筛选（HTS）指标的 Z'因子约为 0.7，表明这是一种出色的筛选系统。这些结果表明，使用 MG 的检测方法非常简单、省力、准确，其通量远远高于现有的 RI 方法。因此，强烈建议该方法在大多数情况下可以取代 RI 方法。这些结果表明它适用于 ACCase 抑制剂的 HTS。

{"title":"Validation of assay for measuring acetyl-coenzyme a carboxylase activity in grasses using malachite green","authors":"Yoshinobu Jin","doi":"10.1016/j.ab.2024.115723","DOIUrl":"10.1016/j.ab.2024.115723","url":null,"abstract":"<div><div>Acetyl-CoA carboxylase (ACCase) is one of the most important enzymes as a herbicide target in gramineous plant species, however, assay methods for the enzyme are primarily limited to those using radioisotopes (RI). Typically, the measurement method that uses RI necessitates specialized facilities and equipment, and involves complex procedures throughout the experiment. As another method for detecting ACCase activity, the colorimetric method using malachite green (MG) is also known. However, reports on this method are limited, and information regarding the simplicity of the procedure and the scope of its application remains unclear. To better understand the method using MG and to develop a simpler assay method, crude enzymes extracted from various target-site resistant (TSR) biotypes of blackgrass (<em>Alopecurus myosuroides</em>) were examined in enzyme inhibition tests. As a result, this method was able to accurately detect the relationship between the chemical classes of ACC inhibitors and cross-resistance to specific TSRs. Moreover, the ACCase activity of other grass species was also examined using this method. By using crude enzymes from various species and a commercially available phosphatase kit containing MG, ACCase activity was detectable with good accuracy. In addition, enzyme inhibition studies using ACCase inhibiting herbicides revealed that this method reproduced results similar to those obtained with the RI method. The Z′-factor, an index of high-throughput screening (HTS), was around 0.7, indicating that it is an excellent screening system. These results suggest that the assay method using MG is very simple, labor-saving, and accurate with a throughput much higher than that of the existing RI method. Therefore, it is strongly suggested that the method could replace the RI method in most cases. These results indicate that it is applicable to HTS for ACCase inhibitors.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115723"},"PeriodicalIF":2.6,"publicationDate":"2024-11-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142715228","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

Sample loading in gel electrophoresis using adapted 3D printers 使用适配的 3D 打印机在凝胶电泳中装载样品。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-11-23 DOI: 10.1016/j.ab.2024.115721

Tao Tao , Alexey Kopyl , Yuki Yew , Fawaz El-Dani , Hassan Ali Abid , Thomas Hiscox , Oi Wah Liew , Tuck Wah Ng

In gel electrophoresis, samples that are dispensed too high above or too low into the wells result in sub-optimal outcomes. Here, an adapted 3D printer liquid handler equipped with an optical sensor was found to attain vertical sample delivery positionings at a standard deviation over mean ratio of 0.008. This illustrated high accuracy and repeatability outcomes achieved using automation that is cost effective and low in technical knowledge demand.

在凝胶电泳过程中，如果将样品分装得过高或过低，都会导致电泳结果不理想。在这里，我们发现，配备光学传感器的改装 3D 打印机液体处理装置可实现垂直样品输送定位，其标准偏差与平均值之比为 0.008。这说明使用自动化技术可以实现高精度和可重复性的结果，而且成本低、技术知识需求少。

引用次数: 0

Studying C9orf72 dipeptide repeat polypeptide aggregation using an analytical ultracentrifuge equipped with fluorescence detection 利用配备荧光检测装置的分析型超速离心机研究 C9orf72 二肽重复多肽的聚集情况

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-11-22 DOI: 10.1016/j.ab.2024.115720

Bashkim Kokona , Nicole R. Cunningham , Jeanne M. Quinn , Danielle R. Jacobsen , F. Jay Garcia , Sierra M. Galindo , Leonard Petrucelli , Walter F. Stafford , Thomas M. Laue , Robert Fairman

Sedimentation velocity, using an analytical ultracentrifuge equipped with fluorescence detection, and electrophoresis methods are used to study aggregation of proteins in transgenic animal model systems. Our previous work validated the power of this approach in an analysis of mutant huntingtin aggregation. We demonstrate that this method can be applied to another neurodegenerative disease studying the aggregation of three dipeptide repeats (DPRs) produced by aberrant translation of mutant c9orf72 containing large G₄C₂ hexanucleotide repeats. These repeat expansions are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We analyzed the aggregation patterns of (Gly-Pro)₄₇, (Gly-Ala)₅₀, and (Gly-Arg)₅₀ fused to fluorescent proteins in samples prepared from D. melanogaster, and (Gly-Ala)₅₀ in C. elegans, using AU-FDS and SDD-AGE. Results suggest that (GP)₄₇ is largely monomeric. In contrast, (GA)₅₀ forms both intermediate and large-scale aggregates. (GR)₅₀ is partially monomeric with some aggregation noted in SDD-AGE analysis. The aggregation of this DPR is likely to represent co-aggregated states with DNA and/or RNA. The power of these methods is the ability to gather data on aggregation patterns and characteristics in animal model systems, which may then be used to interpret the mitigation of aggregation through genetic or molecular therapeutic interventions.

利用配备荧光检测装置的分析超速离心机的沉降速度和电泳方法来研究转基因动物模型系统中蛋白质的聚集。我们之前的工作验证了这种方法在分析突变体亨廷蛋白聚集中的威力。我们证明了这种方法可以应用于另一种神经退行性疾病，研究含有大型 G4C2 六核苷酸重复序列的突变体 c9orf72 的异常翻译产生的三个二肽重复序列（DPRs）的聚集。这些重复序列的扩展是导致家族性肌萎缩侧索硬化症（ALS）和额颞叶痴呆症（FTD）的最常见原因。我们使用 AU-FDS 和 SDD-AGE 分析了黑腹蝇蛹样本中与荧光蛋白融合的 (Gly-Pro)47、(Gly-Ala)50 和 (Gly-Arg)50 的聚集模式，以及秀丽隐杆线虫样本中的 (Gly-Ala)50。结果表明，(GP)47 在很大程度上是单体。相比之下，(GA)50 既能形成中间聚集体，也能形成大规模聚集体。在 SDD-AGE 分析中，(GR)50 部分是单体，但也有一些聚集。这种 DPR 的聚集可能代表与 DNA 和/或 RNA 的共聚集状态。这些方法的优势在于能够收集动物模型系统中有关聚集模式和特征的数据，然后可用于解释通过基因或分子治疗干预来缓解聚集的情况。

{"title":"Studying C9orf72 dipeptide repeat polypeptide aggregation using an analytical ultracentrifuge equipped with fluorescence detection","authors":"Bashkim Kokona , Nicole R. Cunningham , Jeanne M. Quinn , Danielle R. Jacobsen , F. Jay Garcia , Sierra M. Galindo , Leonard Petrucelli , Walter F. Stafford , Thomas M. Laue , Robert Fairman","doi":"10.1016/j.ab.2024.115720","DOIUrl":"10.1016/j.ab.2024.115720","url":null,"abstract":"<div><div>Sedimentation velocity, using an analytical ultracentrifuge equipped with fluorescence detection, and electrophoresis methods are used to study aggregation of proteins in transgenic animal model systems. Our previous work validated the power of this approach in an analysis of mutant huntingtin aggregation<em>.</em> We demonstrate that this method can be applied to another neurodegenerative disease studying the aggregation of three dipeptide repeats (DPRs) produced by aberrant translation of mutant <em>c9orf72</em> containing large G<sub>4</sub>C<sub>2</sub> hexanucleotide repeats<em>.</em> These repeat expansions are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We analyzed the aggregation patterns of (Gly-Pro)<sub>47</sub>, (Gly-Ala)<sub>50</sub>, and (Gly-Arg)<sub>50</sub> fused to fluorescent proteins in samples prepared from <em>D. melanogaster</em>, and (Gly-Ala)<sub>50</sub> in <em>C. elegans</em>, using AU-FDS and SDD-AGE<em>.</em> Results suggest that (GP)<sub>47</sub> is largely monomeric. In contrast, (GA)<sub>50</sub> forms both intermediate and large-scale aggregates. (GR)<sub>50</sub> is partially monomeric with some aggregation noted in SDD-AGE analysis. The aggregation of this DPR is likely to represent co-aggregated states with DNA and/or RNA. The power of these methods is the ability to gather data on aggregation patterns and characteristics in animal model systems, which may then be used to interpret the mitigation of aggregation through genetic or molecular therapeutic interventions.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115720"},"PeriodicalIF":2.6,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142705120","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

Chemiluminescent heterogeneous and homogeneous-heterogeneous assays for determination of nicking endonuclease activity 用于测定核酸内切酶活性的化学发光异构和同构异构测定法。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-11-21 DOI: 10.1016/j.ab.2024.115719

Petr A. Bai , Anton M. Solovjev , Elena A. Kubareva , Sergey A. Kurzeev , Ivan Yu Sakharov

Homogeneous-heterogeneous and heterogeneous formats of a simple and sensitive assay for the determination of nicking endonuclease (NE) Nt.Bst9I activity was developed. The duplex of two 26-membered biotinylated DNA oligonucleotides was used as a substrate of Nt.Bst9I. To improve the assay sensitivity the chemiluminescent detection system based on the use of conjugate of streptavidin and polyperoxidase and enhanced chemiluminescence reaction was used. Both proposed assay formats were constructed using microtiter plates as a solid support, allowing for easy automation of NE analysis using ELISA equipment. Varying the acidity, concentration of KCl and NaCl, and temperature of the reaction medium, favorable conditions were found. Although both formats of the proposed assay can be applied to estimate Nt.Bst9I activity, the heterogenous assay was more sensitive than the homogeneous-heterogeneous one.

开发了一种简单灵敏的测定核酸内切酶（NE）Nt.Bst9I活性的同质-异质和异质检测方法。两个 26 元生物素化 DNA 寡核苷酸的双链体被用作 Nt.Bst9I 的底物。为了提高检测灵敏度，使用了基于链霉亲和素和多过氧化物酶共轭物的化学发光检测系统和增强化学发光反应。所提出的两种检测方法均以微孔板作为固体支持物，从而便于使用 ELISA 设备对 NE 进行自动化分析。通过改变反应介质的酸度、KCl 和 NaCl 的浓度以及温度，找到了有利的条件。尽管所提议的两种检测方法都可用于评估 Nt.Bst9I 的活性，但异质检测方法比同质异构检测方法更灵敏。

引用次数: 0

A biocompatible fluorescent probe for endogenous hydrogen sulfide detection and imaging 用于内源性硫化氢检测和成像的生物相容性荧光探针。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-11-15 DOI: 10.1016/j.ab.2024.115718

Xitian Zhu , Huijia Chen , Fang Ke

Hydrogen sulfide (H₂S) acts as a messenger molecule and can mediate a variety of physiological functions. Conventional methods are seldom used to detect endogenous H₂S and present some difficulties in selective and accurate detection. Reaction-based recognition of endogenous H₂S by organic small molecule probes with good specificity and biocompatibility. To address this challenge, we developed a novel H₂S fluorescent probe 4-(2-(6-hydroxy-2-naphthyl) ethyl)-1-methylpyridinium (DSNP) that triggers a thiolysis reaction through a strong electron withdrawing group, releasing a fluorescent molecule. The simple probe DSNP not only have good selectivity, large Stokes shifts and biocompatibility, but also demonstrated a detection limit as low as 28.4 nM and reaction times as quick as 30 min. Moreover, it has been successfully applied to imaging intracellular H₂S in myeloma cells and zebrafish. This study opens new insights to help push this probe forward for its applicability for detailed H₂S localization studies in osteosarcoma.

硫化氢（H2S）是一种信使分子，可介导多种生理功能。传统方法很少用于检测内源性 H2S，而且在选择性和准确检测方面存在一些困难。基于反应的有机小分子探针识别内源性 H2S 具有良好的特异性和生物相容性。为了应对这一挑战，我们开发了一种新型 H2S 荧光探针 4-(2-(6-羟基-2-萘基)乙基)-1-甲基吡啶鎓（DSNP），它能通过一个强取电子基团引发硫解反应，释放出一个荧光分子。简单的探针 DSNP 不仅具有良好的选择性、较大的斯托克斯位移和生物相容性，而且检测限低至 28.4 nM，反应时间短至 30 分钟。此外，它还被成功应用于骨髓瘤细胞和斑马鱼细胞内 H2S 的成像。这项研究开启了新的视角，有助于推动该探针在骨肉瘤 H2S 定位详细研究中的应用。

{"title":"A biocompatible fluorescent probe for endogenous hydrogen sulfide detection and imaging","authors":"Xitian Zhu , Huijia Chen , Fang Ke","doi":"10.1016/j.ab.2024.115718","DOIUrl":"10.1016/j.ab.2024.115718","url":null,"abstract":"<div><div>Hydrogen sulfide (H<sub>2</sub>S) acts as a messenger molecule and can mediate a variety of physiological functions. Conventional methods are seldom used to detect endogenous H<sub>2</sub>S and present some difficulties in selective and accurate detection. Reaction-based recognition of endogenous H<sub>2</sub>S by organic small molecule probes with good specificity and biocompatibility. To address this challenge, we developed a novel H<sub>2</sub>S fluorescent probe 4-(2-(6-hydroxy-2-naphthyl) ethyl)-1-methylpyridinium (<strong>DSNP</strong>) that triggers a thiolysis reaction through a strong electron withdrawing group, releasing a fluorescent molecule. The simple probe <strong>DSNP</strong> not only have good selectivity, large Stokes shifts and biocompatibility, but also demonstrated a detection limit as low as 28.4 nM and reaction times as quick as 30 min. Moreover, it has been successfully applied to imaging intracellular H<sub>2</sub>S in myeloma cells and zebrafish. This study opens new insights to help push this probe forward for its applicability for detailed H<sub>2</sub>S localization studies in osteosarcoma.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115718"},"PeriodicalIF":2.6,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142643262","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 novel colorimetric assay for the detection of urinary N1, N12-diacetylspermine, a known biomarker for colorectal cancer 用于检测尿液中 N1、N12-二乙酰精胺（一种已知的结直肠癌生物标记物）的新型比色测定法。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-11-12 DOI: 10.1016/j.ab.2024.115717

Dipanjan Bhattacharyya , Marcia A. LeVatte , Upasana Singh , Fleur Issac , Mahmoud Karim , Saira Ali , August Sieben , Suyenna Huang , David S. Wishart

Urinary N¹, N¹²-diacetylspermine (DAS) is a known biomarker for colorectal cancer (CRC). However, DAS levels in both healthy and CRC patients’ urine samples are extremely low and often challenging to quantify. Complex and expensive methods do exist to detect DAS in urine, but simpler, less expensive methods to detect DAS are needed, especially in low resource settings. Here we describe a highly efficient, fast, precise, and inexpensive colorimetric assay to detect low levels of DAS in human urine samples. We used recombinant diacetylspermine oxidase (rDAS Ox), expressed and extracted from E. coli, to oxidize DAS, producing three products including hydrogen peroxide (H₂O₂). The level of DAS present, which correlates with H₂O₂ levels, was measured using horseradish peroxidase (HRP), which together with H₂O₂, oxidized Amplex™ Red to produce the pink-colored resorufin. The concentration of resorufin is directly proportional to H₂O₂ (and DAS) levels. As urine contains metabolites which interfere with these oxidation reactions, we developed a simple two column-based protocol using ion exchange resins to remove these compounds and concentrate the DAS. With this novel cleaning and concentrating method, DAS was concentrated 15 times (confirmed by nuclear magnetic resonance (NMR) spectroscopy) and <1 μM DAS could be detected. Correlation graphs of urine samples spiked with known DAS concentrations versus assay-determined DAS concentrations had high coefficients of determination (R²) for 0–10 μM DAS (0.94) and for 0–1 μM DAS (0.91), clearly demonstrating the excellent performance of the two-column protocol with the rDAS Ox reaction mixture. To the best of our knowledge, this is first reported colorimetric enzymatic assay that quantitates DAS in urine.

尿液中的 N1、N12-二乙酰精胺（DAS）是已知的结直肠癌（CRC）生物标志物。然而，健康尿样和 CRC 患者尿样中的 DAS 含量都极低，而且通常难以量化。检测尿液中 DAS 的方法复杂且昂贵，但我们需要更简单、更便宜的方法来检测 DAS，尤其是在资源匮乏的环境中。在此，我们介绍了一种高效、快速、精确且廉价的比色法，用于检测人体尿样中低水平的 DAS。我们使用从大肠杆菌中表达和提取的重组双乙酰精胺氧化酶（rDAS Ox）来氧化 DAS，产生包括过氧化氢（H2O2）在内的三种产物。辣根过氧化物酶（HRP）与 H2O2 一起氧化 Amplex™ Red，生成粉红色的 resorufin，DAS 的含量与 H2O2 的含量相关。雷索卢芬的浓度与 H2O2（和 DAS）水平成正比。由于尿液中含有干扰这些氧化反应的代谢物，我们开发了一种简单的双柱方案，使用离子交换树脂去除这些化合物并浓缩 DAS。通过这种新颖的清洁和浓缩方法，DAS 浓缩了 15 倍（经核磁共振 (NMR) 光谱证实），2) 0-10 μM DAS 的浓缩率为 0.94，0-1 μM DAS 的浓缩率为 0.91，这清楚地表明了双柱方案与 rDAS Ox 反应混合物的卓越性能。据我们所知，这是首次报道定量检测尿液中 DAS 的比色酶法。

{"title":"A novel colorimetric assay for the detection of urinary N1, N12-diacetylspermine, a known biomarker for colorectal cancer","authors":"Dipanjan Bhattacharyya , Marcia A. LeVatte , Upasana Singh , Fleur Issac , Mahmoud Karim , Saira Ali , August Sieben , Suyenna Huang , David S. Wishart","doi":"10.1016/j.ab.2024.115717","DOIUrl":"10.1016/j.ab.2024.115717","url":null,"abstract":"<div><div>Urinary N<sup>1</sup>, N<sup>12</sup>-diacetylspermine (DAS) is a known biomarker for colorectal cancer (CRC). However, DAS levels in both healthy and CRC patients’ urine samples are extremely low and often challenging to quantify. Complex and expensive methods do exist to detect DAS in urine, but simpler, less expensive methods to detect DAS are needed, especially in low resource settings. Here we describe a highly efficient, fast, precise, and inexpensive colorimetric assay to detect low levels of DAS in human urine samples. We used recombinant diacetylspermine oxidase (rDAS Ox), expressed and extracted from <em>E. coli</em>, to oxidize DAS, producing three products including hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>). The level of DAS present, which correlates with H<sub>2</sub>O<sub>2</sub> levels, was measured using horseradish peroxidase (HRP), which together with H<sub>2</sub>O<sub>2</sub>, oxidized Amplex™ Red to produce the pink-colored resorufin. The concentration of resorufin is directly proportional to H<sub>2</sub>O<sub>2</sub> (and DAS) levels. As urine contains metabolites which interfere with these oxidation reactions, we developed a simple two column-based protocol using ion exchange resins to remove these compounds and concentrate the DAS. With this novel cleaning and concentrating method, DAS was concentrated 15 times (confirmed by nuclear magnetic resonance (NMR) spectroscopy) and <1 μM DAS could be detected. Correlation graphs of urine samples spiked with known DAS concentrations versus assay-determined DAS concentrations had high coefficients of determination (R<sup>2</sup>) for 0–10 μM DAS (0.94) and for 0–1 μM DAS (0.91), clearly demonstrating the excellent performance of the two-column protocol with the rDAS Ox reaction mixture. To the best of our knowledge, this is first reported colorimetric enzymatic assay that quantitates DAS in urine.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115717"},"PeriodicalIF":2.6,"publicationDate":"2024-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142612401","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Assays to measure small molecule Hsp70 agonist activity in vitro and in vivo 测量小分子 Hsp70 激动剂体外和体内活性的试验。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-11-09 DOI: 10.1016/j.ab.2024.115712

Olivia Shapiro , Clara Woods , Amanda M. Gleixner , Sara Sannino , Marilyn Ngo , Michael D. McDaniels , Peter Wipf , Neil A. Hukriede , Christopher J. Donnelly , Jeffrey L. Brodsky

Hsp70 prevents protein aggregation and is cytoprotective, but sustained Hsp70 overexpression is problematic. Therefore, we characterized small molecule agonists that augment Hsp70 activity. Because cumbersome assays were required to assay agonists, we developed cell-based and in vivo assays in which disease-associated consequences of Hsp70 activation can be quantified. One assay uses an optogenetic system in which the formation of TDP-43 inclusions can be controlled, and the second assay employs a zebrafish model for acute kidney injury (AKI). These complementary assays will facilitate future work to identify new Hsp70 agonists as well as optimized agonist derivatives.

Hsp70 可防止蛋白质聚集并具有细胞保护作用，但持续过量表达 Hsp70 会带来问题。因此，我们开发了能增强 Hsp70 活性的小分子激动剂。由于检测激动剂需要进行繁琐的试验，我们开发了基于细胞的试验和体内试验，通过这些试验可以量化 Hsp70 激活对疾病造成的影响。其中一种试验使用光遗传系统，该系统可控制 TDP-43 包涵体的形成；第二种试验使用斑马鱼急性肾损伤（AKI）模型。这些互补性试验将有助于未来鉴定新的 Hsp70 激动剂以及其他优化的 MAL1-271 衍生物的工作。

引用次数: 0

Development of an assay to quantify tranexamic acid levels in plasma 开发一种测定血浆中氨甲环酸水平的方法。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-11-08 DOI: 10.1016/j.ab.2024.115714

Paul Y. Kim , Michelle Vong , Dani Lee , Chengliang Wu

Dysregulations of blood clot breakdown (fibrinolysis) during vascular trauma can lead to excessive blood loss. Tranexamic acid (TXA) is an inhibitor of fibrinolysis that works by blocking the interaction between plasminogen and fibrin degradation products (FDPs) – a key step in fibrinolysis. Despite the widespread usage, there are no tests available in a clinical setting to monitor TXA levels. We developed a fluorescence resonance energy transfer (FRET)-based assay to quantify TXA concentrations in plasma by using 1) fluorescently labeled plasminogen, and 2) FDPs labeled with a fluorescence quencher. Once plasminogen binds the FDPs, the fluorescent signal is quenched. TXA causes plasminogen to dissociate from the FDPs, thus increasing fluorescence signal in a dose-dependent manner. The dose response was sensitive between 1 and 100 μM (0.16 and 15.7 mg/L). The intraassay and interassay variabilities were determined to be 5.7 % and 3.0 %, respectively. Limit of detection was estimated to be 0.28 μM (0.044 mg/L). When tested for measuring known levels of TXA added to plasma samples, the ratio between measured and expected TXA concentration was 1.0151. Our study demonstrates a novel assay that can rapidly quantify TXA concentrations in plasma samples, thus demonstrating its potential as an in-hospital tool.

血管创伤期间血凝块分解（纤溶）失调会导致失血过多。氨甲环酸（TXA）是一种纤维蛋白溶解抑制剂，通过阻断纤溶过程中的关键步骤--纤溶酶原与纤维蛋白降解产物（FDPs）之间的相互作用而发挥作用。尽管TXA被广泛使用，但目前临床上还没有可用来监测TXA水平的检测方法。我们开发了一种基于荧光共振能量转移（FRET）的检测方法，通过使用 1）荧光标记的纤溶酶原和 2）荧光淬灭剂标记的 FDPs 来量化血浆中的 TXA 浓度。一旦纤溶酶原与 FDPs 结合，荧光信号就会被淬灭。TXA 会导致纤溶酶原与 FDPs 分离，从而以剂量依赖的方式增加荧光信号。剂量反应灵敏度在 1 到 100 μM（0.16 到 15.7 mg/L）之间。测定内和测定间变异性分别为 5.7 % 和 3.0 %。检测限估计为 0.28 μM（0.044 mg/L）。当测试测量血浆样本中添加的已知 TXA 水平时，测量值与预期 TXA 浓度的比值为 1.0151。我们的研究展示了一种可快速定量血浆样本中 TXA 浓度的新型检测方法，从而证明了其作为院内工具的潜力。

{"title":"Development of an assay to quantify tranexamic acid levels in plasma","authors":"Paul Y. Kim , Michelle Vong , Dani Lee , Chengliang Wu","doi":"10.1016/j.ab.2024.115714","DOIUrl":"10.1016/j.ab.2024.115714","url":null,"abstract":"<div><div>Dysregulations of blood clot breakdown (fibrinolysis) during vascular trauma can lead to excessive blood loss. Tranexamic acid (TXA) is an inhibitor of fibrinolysis that works by blocking the interaction between plasminogen and fibrin degradation products (FDPs) – a key step in fibrinolysis. Despite the widespread usage, there are no tests available in a clinical setting to monitor TXA levels. We developed a fluorescence resonance energy transfer (FRET)-based assay to quantify TXA concentrations in plasma by using 1) fluorescently labeled plasminogen, and 2) FDPs labeled with a fluorescence quencher. Once plasminogen binds the FDPs, the fluorescent signal is quenched. TXA causes plasminogen to dissociate from the FDPs, thus increasing fluorescence signal in a dose-dependent manner. The dose response was sensitive between 1 and 100 μM (0.16 and 15.7 mg/L). The intraassay and interassay variabilities were determined to be 5.7 % and 3.0 %, respectively. Limit of detection was estimated to be 0.28 μM (0.044 mg/L). When tested for measuring known levels of TXA added to plasma samples, the ratio between measured and expected TXA concentration was 1.0151. Our study demonstrates a novel assay that can rapidly quantify TXA concentrations in plasma samples, thus demonstrating its potential as an in-hospital tool.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115714"},"PeriodicalIF":2.6,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142612441","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A simple and enzyme-free method for sensitive p53 analysis based on DNAzyme-mediated signal amplification 一种基于 DNA 酶介导的信号放大的简单、无酶的 p53 敏感分析方法。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-11-08 DOI: 10.1016/j.ab.2024.115716

Jia Deng , Ye Yuan , Min Zou , Xudong Liu , Xianxian Zhao , Hongli Liu

There is an urgent demand for a simple yet extremely accurate biosensor to analyze tumorigenesis. Herein, we present a novel fluorescent and enzyme-free approach for detecting p53 gene cascading proximity ligation-mediated catalytic hairpin assembly and DNAzyme-assisted signal reaction. When the target p53 gene is present, the interaction between p53 and L1 and L2 chains initiates catalytic hairpin assembly and subsequently exposes DNAzyme in the P3 probe. The exposed DNAzyme binds with the loop region of the P4 probe and generates a nicking site, resulting in the release of a significant amount of ATMND that is conjugated in the stem section of P4. This leads to an amplified fluorescence response, which serves as a fluorescence signal for the detection of the p53 gene. This method allows for the accurate and sensitive identification of the p53 gene, exhibiting a linear reaction range of 1 fM to 1 nM, with a limit of detection as low as 0.23 fM. Furthermore, this fluorescent method has been utilized for the examination of clinical samples with a favorable recovery rate. Crucially, this versatile platform may be expanded to analyze different targets by changing the corresponding recognition unit, showing great potential for point-of-care testing in tumorigenesis analysis.

目前迫切需要一种简单而又极其精确的生物传感器来分析肿瘤的发生。在此，我们提出了一种新型的荧光和无酶方法，用于检测 p53 基因级联近接连接介导的催化发夹组装和 DNA 酶辅助的信号反应。当目标 p53 基因存在时，p53 与 L1 和 L2 链之间的相互作用会启动催化发夹组装，并随后暴露 P3 探针中的 DNA 酶。暴露的 DNA 酶与 P4 探针的环状区域结合，产生一个挑刺位点，从而释放出大量与 P4 茎部结合的 ATMND。这导致荧光反应放大，成为检测 p53 基因的荧光信号。这种方法可以准确灵敏地鉴定 p53 基因，其线性反应范围为 1 fM 至 1 nM，检测限低至 0.23 fM。此外，这种荧光方法还可用于临床样本的检测，并具有良好的回收率。最重要的是，这种多功能平台可以通过改变相应的识别单元来分析不同的靶标，在肿瘤发生分析的床旁检测方面显示出巨大的潜力。

{"title":"A simple and enzyme-free method for sensitive p53 analysis based on DNAzyme-mediated signal amplification","authors":"Jia Deng , Ye Yuan , Min Zou , Xudong Liu , Xianxian Zhao , Hongli Liu","doi":"10.1016/j.ab.2024.115716","DOIUrl":"10.1016/j.ab.2024.115716","url":null,"abstract":"<div><div>There is an urgent demand for a simple yet extremely accurate biosensor to analyze tumorigenesis. Herein, we present a novel fluorescent and enzyme-free approach for detecting p53 gene cascading proximity ligation-mediated catalytic hairpin assembly and DNAzyme-assisted signal reaction. When the target p53 gene is present, the interaction between p53 and L1 and L2 chains initiates catalytic hairpin assembly and subsequently exposes DNAzyme in the P3 probe. The exposed DNAzyme binds with the loop region of the P4 probe and generates a nicking site, resulting in the release of a significant amount of ATMND that is conjugated in the stem section of P4. This leads to an amplified fluorescence response, which serves as a fluorescence signal for the detection of the p53 gene. This method allows for the accurate and sensitive identification of the p53 gene, exhibiting a linear reaction range of 1 fM to 1 nM, with a limit of detection as low as 0.23 fM. Furthermore, this fluorescent method has been utilized for the examination of clinical samples with a favorable recovery rate. Crucially, this versatile platform may be expanded to analyze different targets by changing the corresponding recognition unit, showing great potential for point-of-care testing in tumorigenesis analysis.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115716"},"PeriodicalIF":2.6,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142612419","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

Specialized greenness sustainability tools for evaluation of the spectrophotometric methodologies greenness: Spectral signal manipulation for resolving the interfering telmisartan and metoprolol succinate spectra in their bulk and pharmaceutical formulation 评估分光光度法绿色可持续性的专用工具：光谱信号操作，用于消除替米沙坦和琥珀酸美托洛尔散装和药物制剂中的干扰光谱。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2024-11-08 DOI: 10.1016/j.ab.2024.115711

Michael Gamal Fawzy , Soad S. Abd El-Hay , Alaa Ahmed Mostafa , Youstina Mekhail Metias

Hypertension is a leading cause of cardiovascular mortality, often accompanied by complications such as arrhythmia and stroke. This silent killer requires a multifaceted pharmacological approach for effective management. This article presents new, environmentally friendly spectrophotometric methods for simultaneous quantification of telmisartan (TER) and metoprolol succinate (MTR) in laboratory prepared mixtures and pharmaceutical formulations. The suggested methodologies include the following: area under the curve method (AUC) utilizing area at specific wavelength ranges 228–233 nm (λ₁ – λ₂) and 240–245 nm (λ₃ – λ₄) for each analyte and Fourier self-deconvolution method (FD) depending on built-in function to address spectral interferences. In addition, the induced dual wavelength method (IDWL) employing equality factors to obtain absorbance differences at designated wavelengths, ratio difference method (RD) utilizing divisor-based ratio spectra where the utilized divisors were TER 40 μg/mL and MTR 90 μg/mL, and ratio derivative method (RDV) generating spectra through first derivative application that was measured at 266 nm and 246 nm for TER and MTR, respectively. These methods offer green alternatives for the accurate and precise determination of TER and MTR with exceptional linearity of 3–45, and 15–200 μg/mL for TER and MTR, respectively. Furthermore, the methods showed a coefficient of determination exceeding 0.9995 and good detection and quantification levels. A comprehensive greenness assessment, employing five distinct evaluation tools, confirmed the reduced environmental impact of the proposed methods in terms of waste generation, chemical consumption, and instrument safety. Successful analysis of pharmaceutical formulations and laboratory prepared mixtures containing different TER and MTR ratios confirmed the validity of the proposed methods. Standard addition studies further supported these findings, and the statistical results were comparable to those obtained using a reference method.

高血压是心血管疾病死亡的主要原因，通常伴有心律失常和中风等并发症。要有效控制这一无声杀手，需要采取多方面的药物治疗方法。本文介绍了一种新型、环保的分光光度法，用于同时定量检测实验室制备的混合物和药物制剂中的替米沙坦（TER）和琥珀酸美托洛尔（MTR）。建议采用的方法包括：利用特定波长范围 228-233 nm (λ1 - λ2) 和 240-245 nm (λ3 - λ4) 下的面积对每种分析物进行曲线下面积法 (AUC)，以及傅立叶自旋法 (FD)，根据内置功能解决光谱干扰问题。此外，诱导双波长法 (IDWL) 利用相等因子获得指定波长的吸光度差值；比值差法 (RD) 利用基于除数的比值光谱，其中使用的除数为 TER 40 μg/mL 和 MTR 90 μg/mL；比值导数法 (RDV) 通过一阶导数应用生成光谱，分别在 266 nm 和 246 nm 处测量 TER 和 MTR。这些方法为准确和精确地测定 TER 和 MTR 提供了绿色的替代方法，TER 和 MTR 的线性范围分别为 3-45 和 15-200 μg/mL。此外，这些方法的测定系数超过 0.9995，检测和定量水平良好。利用五种不同的评估工具进行的综合绿色评估证实，所建议的方法在废物产生、化学品消耗和仪器安全方面减少了对环境的影响。对含有不同 TER 和 MTR 比率的药物制剂和实验室制备的混合物的成功分析证实了建议方法的有效性。标准添加研究进一步支持了这些发现，统计结果与使用参考方法获得的结果相当。

{"title":"Specialized greenness sustainability tools for evaluation of the spectrophotometric methodologies greenness: Spectral signal manipulation for resolving the interfering telmisartan and metoprolol succinate spectra in their bulk and pharmaceutical formulation","authors":"Michael Gamal Fawzy , Soad S. Abd El-Hay , Alaa Ahmed Mostafa , Youstina Mekhail Metias","doi":"10.1016/j.ab.2024.115711","DOIUrl":"10.1016/j.ab.2024.115711","url":null,"abstract":"<div><div>Hypertension is a leading cause of cardiovascular mortality, often accompanied by complications such as arrhythmia and stroke. This silent killer requires a multifaceted pharmacological approach for effective management. This article presents new, environmentally friendly spectrophotometric methods for simultaneous quantification of telmisartan (TER) and metoprolol succinate (MTR) in laboratory prepared mixtures and pharmaceutical formulations. The suggested methodologies include the following: area under the curve method (AUC) utilizing area at specific wavelength ranges 228–233 nm (<em>λ</em><sub>1</sub> – <em>λ</em><sub>2</sub>) and 240–245 nm (<em>λ</em><sub>3</sub> – <em>λ</em><sub>4</sub>) for each analyte and Fourier self-deconvolution method (FD) depending on built-in function to address spectral interferences. In addition, the induced dual wavelength method (IDWL) employing equality factors to obtain absorbance differences at designated wavelengths, ratio difference method (RD) utilizing divisor-based ratio spectra where the utilized divisors were TER 40 μg/mL and MTR 90 μg/mL, and ratio derivative method (RDV) generating spectra through first derivative application that was measured at 266 nm and 246 nm for TER and MTR, respectively. These methods offer green alternatives for the accurate and precise determination of TER and MTR with exceptional linearity of 3–45, and 15–200 μg/mL for TER and MTR, respectively. Furthermore, the methods showed a coefficient of determination exceeding 0.9995 and good detection and quantification levels. A comprehensive greenness assessment, employing five distinct evaluation tools, confirmed the reduced environmental impact of the proposed methods in terms of waste generation, chemical consumption, and instrument safety. Successful analysis of pharmaceutical formulations and laboratory prepared mixtures containing different TER and MTR ratios confirmed the validity of the proposed methods. Standard addition studies further supported these findings, and the statistical results were comparable to those obtained using a reference method.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115711"},"PeriodicalIF":2.6,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142612444","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