A single microcystin in a toxic Microcystis bloom from the river Río de la Plata (Argentina)

Microcystis is one of the most common bloom-forming cyanobacteria genera in diverse ecosystems. More than 80% of its strains are toxic, mainly due to their ability to produce metabolites known as microcystins (MC). Here we report on a M. aeruginosa bloom that appeared in the summer of 2001 at a site of the Río de la Plata, within the city of Buenos Aires. The symptoms in mice indicated that the bloom was hepatotoxic. LC-PDA analysis revealed a similar high concentration (0.9–1?mg?g^–1?d w) of just one MC in the bloom biomass during the 3-month study period. During this period most of the MC (ca. 98 %) was found intracellularly, and the content remained almost the same. The molecular mass of the bloom MC was 1036?Da, as deduced from LC-ESI-MS data. Fragment ion analysis by LC-IT-MS-MS allowed identifying 6 out of the 7 amino acids, as well as their position in the molecule. The molecular mass of the unidentified amino acid residue was 155?Da. According to the data obtained, the MC under study was MC-XR, X representing the unidentified amino acid. This is the first report both on the characterisation of MCs in an urban site of the Río de la Plata waters, and on an Argentinean bloom exhibiting only one MC variant.     

Analysis of microcystins by capillary zone electrophoresis coupling with electrospray ionization mass spectrometry

In this paper, a rapid and effective method based on capillary zone electrophoresis (CZE) coupled with electrospray ionization mass spectrometry (ESI-MS) was established for the trace analysis of microcystin (MC) isomers in crude algae sample. The experimental conditions including the composition, acidity and concentration of buffer, separation voltage, injection time, and MS detection parameters were investigated in detail. A capillary separation system was as follows: a uncoated fused-silica capillary tube (50 μm i.d. × 90 cm), 40 mmol L⁻¹ ammonium acetate solution (pH 9.86) as running buffer, 25 kV as separation voltage, 20 kV × 3 s water first and 20 kV × 20 s for sample injection. Mass analysis was performed in ESI source, with sheath gas temperature 150 °C, sheath gas pressure 10 psi, and sheath gas flow 6 L min⁻¹. And sheath liquid was 7.5 mmol L⁻¹ acetic acid in 50% isopropanol-water (3 μL min⁻¹). Protonation and ammonium adduct molecular ions m/z 506.9 (MC-LR) and 532.0 (MC-YR) were used for the quantification of MCs. Under these conditions, two MCs were baseline separated within 9 min, the calibration curves were obtained in the range of 0.11-10.0 μg mL⁻¹ and 0.16-10.5 μg mL⁻¹ for MC-LR and MC-YR, respectively. Meanwhile, limits of detection were 0.05 and 0.08 μg mL⁻¹ for MC-LR and MC-YR, respectively. The recoveries for the two MCs were in the range of 95.8-108%. The developed approach had been successfully applied to the analysis of MCs in crude algae samples.     

Complete assignment of the NMR spectra of [D‐Leu1]‐microcystin‐LR and analysis of its solution structure

[D ‐Leu¹]‐microcystin‐LR is a recently discovered microcystin. We report the isolation of this microcystin analogue from a Microcystis aeruginosa strain isolated from the Lagoa de Iquipari, Rio de Janeiro, Brazil. The ¹H and ¹³C NMR spectra were completely assigned in both MeOH‐d₄ and DMSO‐d₆. Further, the solution structure of this compound was investigated with the use of two‐dimensional NMR and the amide proton temperature dependence, and was compared with those of its analogs, microcystin‐RR and microcystin‐LR. Copyright © 2002 John Wiley & Sons, Ltd.     

基于HDT自组装金纳米粒子修饰电极的微囊藻毒素-LR电化学免疫传感器研究

构建了一种新型的基于金纳米粒子(Au NPs)修饰金电极的微囊藻毒素-亮氨酸-精氨酸(MCLR)电化学免疫传感器。采用柠檬酸钠还原法制备了Au NPs溶胶,分别用透射电子显微镜和紫外-可见吸收光谱对其进行表征。将Au NPs组装到1,6-己二硫醇(HDT)自组装单分子层修饰的金电极表面,再将MCLR抗体(anti-MCLR)固定于该修饰电极上,利用扫描探针显微镜法、循环伏安法和电化学交流阻抗法(EIS)表征了自制化学修饰电极表面的形貌特征和电化学免疫传感器的电化学特征。通过辣根过氧化物酶标记的MCLR(MCLR-HRP)与MCLR竞争结合抗体,建立了检测MCLR的差分脉冲伏安法(DPV)。在最佳实验条件下,用DPV对MCLR检测的线性范围为0.01~25μg/L,检出限为0.005μg/L。对构建的免疫传感器的重现性、稳定性和选择性进行了考察。该方法对实际水样中MCLR的加标回收率为100%~102%,测定结果与高效液相色谱法的测定结果一致。     

An enhanced LC-MS/MS method for microcystin-LR in lake water

A LC-MS/MS method with enhanced sensitivity and specificity was established for monitoring microcystin-LR (MC-LR) in drinking water supplies in southern Taiwan. The enhanced sensitivity was achieved by the selection of a doubly charged MC-LR as the precursor ion to result in an multiple reaction monitoring (MRM) pair ions of m/z 498.6 --> 135.0. Using this ion pair, a record low detection limit of 2 pg was achieved on column, found in the available literature. A sample preparation method involving C8 solid-phase extraction gave satisfactory recoveries of the analyte. Nodularin, with structural similarity to MC-LR, was used as an internal standard to minimize matrix effects of water samples collected from six different water reservoirs in southern Taiwan, where MC-LR was detected at sub-ppb levels in all the reservoirs. The best precision and accuracy of this method were found with samples prepared to contain MC-LR at 0.1 and 1 microg l(-1). This new method requires considerably smaller water sample volumes because of enhanced quantification sensitivity and hence reduces the time needed for analysis. It should serve as a useful example for method development for monitoring other members of the microcystin family in drinking water supplies.     

基因工程抗体在微囊藻毒素检测分析中的应用研究

微囊藻毒素是蓝藻水华过程中产生的一类小分子多肽生物毒素,能对人和动物的肝脏造成严重损伤,属于2B类致癌物质。由于全球性工业和生活废水的大量排放以及气候变暖等现实因素,蓝藻水华现象日趋频繁和加剧,使得微囊藻毒素在水体和土壤中大量蓄积,严重威胁生态环境和农产品质量安全。基于"抗体-抗原"识别原理的免疫学检测方法是当前生物毒素快速检测研究的热点,近年抗体创制技术突飞猛进,现已从传统的多克隆抗体和单克隆抗体逐渐发展到了新型的基因工程人工抗体创制阶段。该文系统阐述了微囊藻毒素产生的内外因素及其对生态系统、人和动物的主要危害风险;全面梳理了基因工程抗体创制技术及其在微囊藻毒素检测应用研究上的最新进展;并结合作者及所在科研团队最新研究成果,深入探讨了基因工程抗体在微囊藻毒素检测应用上的发展趋势、存在的问题以及拟解决的对策。     

检测产毒微囊藻的特异性探针研究

通过对9株淡水微囊藻的ITS序列及其微囊藻毒素合成酶基因簇(mcy)的分析,以期获得用于快速检测产毒微囊藻的特异性探针.结果表明:9株淡水微囊藻的ITS序列差异百分比达1.0%~5.0%,平均差异度达3.0%,但序列比对得到的差异性片段与其是否产微囊藻毒素之间无明显相关性,无法用于检测产毒微囊藻特异性引物的设计.根据微囊藻毒素合成基因簇的保守结构域设计的6对引物(MCYAAAF\MCYAAAR,MCYAMAF\MCYAMAR,MCYBAF\CYBAR,MEAF\MCYEAR,MCYGAF\MCYGR,MCYKSF\MCYKSR),对9株微囊藻mcy基因簇的扩增进行了研究,结果与LC-MS的检测结果具有很好的一致性.同时,6对引物具有相同的退火温度,可进行PCR的同步扩增,在提高扩增效率的同时也大大增加了检测结果的准确性.此外,根据微囊藻毒素基因筛选设计了用于检测产毒微囊藻的特异性探针.     

Synthesis of Highly Selective Submicromolar Microcystin‐Based Inhibitors of Protein Phosphatase (PP)2A over PP1

Research and therapeutic targeting of the phosphoserine/threonine phosphatases PP1 and PP2A is hindered by the lack of selective inhibitors. The microcystin (MC) natural toxins target both phosphatases with equal potency, and their complex synthesis has complicated structure–activity relationship studies in the past. We report herein the synthesis and biochemical evaluation of 11 MC analogues, which was accomplished through an efficient strategy combining solid‐ and solution‐phase approaches. Our approach led to the first MC analogue with submicromolar inhibitory potency that is strongly selective for PP2A over PP1 and does not require the complex lipophilic Adda group. Through mutational and structural analyses, we identified a new key element for binding, as well as reasons for the selectivity. This work gives unprecedented insight into how selectivity between these phosphatases can be achieved with MC analogues.     

液相色谱-二极管阵列检测器/离子阱质谱法检测水中的5种微囊藻毒素

建立了一种液相色谱-二极管阵列检测器(LC-DAD)/离子阱质谱(IT MS)对水中5种微囊藻毒素(microcystins)的分析方法。水中的微囊藻毒素经固相萃取富集和净化,经LC分离后,采用DAD和IT MS定性分析,DAD定量分析。在优化的条件下,水中5种微囊藻毒素的检出限为0.1 μg/L; 3个质量浓度加标水平(0.2、0.8和5 μg/L)的平均回收率为52.2%～115.2%,相对标准偏差为1.2%～10.0%。该方法从紫外吸收光谱和质谱角度同时进行定性定量分析,可用于地表水和饮用水中多种微囊藻毒素的检测。