基于纳米磁珠和双标记抗体的玉米赤霉烯酮快速、高灵敏检测方法的建立及应用

玉米赤霉烯酮(zeralenone,ZEN)具有雌激素活性,主要污染谷物和饲料,大量聚积可导致流产和死胎,给动物和人类健康带来严重威胁。本研究通过将ZEN偶联抗原ZEN-BSA包被于纳米磁珠(magnetic nanoparticles,MNPs),制备纳米磁珠-偶联抗原复合物(MNPs-BSA-ZEN),同时使用金颗粒(Au nanoparticles,AuNPs)和辣根过氧化物酶(horseradish peroxidase,HRP)双标记的ZEN单克隆抗体,建立新型酶联免疫检测方法(MNPs-HRP-AuNPsIC-ELISA)。检测下限(IC10)达到0.03ng/mL,检测区间(IC20–IC80)为0.05–0.89ng/mL,半数抑制率(IC50)为0.22ng/mL,与ZEN类似物(α-zearalanol、zearalanone、α-zearalenol、β-zearalenol和β-zearalanol)的交叉反应性依次为19.2%、11.7%、8.3%、1.2%和4.3%,与黄曲霉毒素B1、赭曲霉毒素A、伏马毒素B1、桔青霉素和展青霉毒素几乎不存在交叉反应。在玉米、面粉和大豆样本中的加标回收率可达81.6%–113.5%,与LC-MS/MS同时对天然样本中ZEN含量的检测结果表明,两种方法相关性良好。本研究建立的MNPs-HRP-AuNPs IC-ELISA具备快速和高灵敏的双重优势,也可为其他霉菌毒素精准检测技术的开发提供参考。     

基于纳米金和辣根过氧化物酶双标记抗体的黄曲霉毒素B1高灵敏检测方法的建立及应用

黄曲霉毒素B₁（aflatoxin B₁,AFB₁）在自然界普遍存在,可污染多种粮食作物和饲料,给动物和人类健康造成严重威胁。为建立AFB₁高灵敏度的快速检测方法,本研究通过采用纳米金颗粒（Au nanoparticles,AuNPs）和辣根过氧化物酶（horseradish peroxidase,HRP）双标记AFB₁单克隆抗体,建立新型酶联免疫检测方法（HRP-AuNPs IC-ELISA）,检测下限（IC₁₀）为0.017ng/mL,检测区间（IC₂₀-IC₈₀）为0.026-0.376ng/mL,半数抑制率（IC₅₀）为0.099ng/mL,与黄曲霉毒素B₂、G₁、G₂ 和M₁ 的交叉反应率分别为2.7%、9.3%、2.1%和5.3%,与赭曲霉毒素A、伏马毒素B₁、桔青霉素、展青霉毒素和玉米赤霉烯酮几乎不存在交叉反应。在玉米和面粉样本中的加标回收率可达88.93-103.55%,与LC-MS/MS同时对天然样本中AFB₁ 含量进行检测,结果表明,两种方法相关性良好。本研究建立的HRP-AuNPs IC-ELISA耗时短且灵敏度高,可用于实际样本中AFB₁ 的快速定量检测与分析,也为其他霉菌毒素的精准检测技术开发提供参考。     

基于量子点标记的赭曲霉毒素A快速、高灵敏荧光免疫层析检测方法的建立及应用

赭曲霉毒素A（ochratoxin A,OTA）具有肾毒性、致畸性、致癌性和免疫毒性,广泛存在于各种粮食作物及其副产品中,是食品和饲料原料的重要污染物,可在人类及动物体内蓄积,在已知发现的真菌毒素中,重要性和危害性仅次于黄曲霉毒素。本研究通过采用量子点荧光微球（quantum dots,QDs）标记OTA单克隆抗体,并基于免疫层析原理,优化、建立了OTA高灵敏荧光免疫层析检测方法（FICGA）,15min即可实现对农产品中OTA污染的快速定量检测。该方法检测下限（IC₁₀）达到0.04ng/mL,检测区间（IC₂₀-IC₈₀）为0.05-0.59ng/mL,半数抑制率（IC₅₀）为0.18ng/mL。与OTA类似物OTB、OTC交叉反应性为7.3%和11.9%,对其他常见真菌毒素AFB₁、ZEN、FB₁和DON均无交叉反应。在玉米、面粉和大豆样本中的加标回收率可达83.2%-117.8%,与LC-MS/MS同时对天然样本中OTA含量的检测结果表明,两种方法相关性良好。本研究建立的FICGA快速、灵敏,可满足基层单位和现场的快速检测需求,具有很好的应用前景。     

稳定表达人源GABAAR-CHO细胞株的建立

目的: 构建α₁亚基诱导表达、β₂和γ_2L亚基稳定表达的人源α₁β₂γ_2L-GABA_AR-CHO(Chinese hamster ovary)细胞株。方法: 从人cDNA文库中扩增α₁、β₂、γ_2L亚基编码基因,分别构建亚基表达载体;将三个亚基表达载体共转染CHO-K1细胞,通过抗性筛选、膜电位检测法进行稳定表达克隆筛选;通过qPCR、Western blot对亚基表达进行鉴定;以激动剂GABA、阳性变构调节剂地西泮(diazepam,Dia)、拮抗剂荷包牡丹碱(bicuculine)为工具药,采用全细胞膜片钳方法及膜电位检测法对稳定表达细胞的药理学功能进行鉴定。结果: 经克隆筛选获得表达量较高的α₁β₂γ_2L-GABA_AR-CHO并对其亚基表达鉴定,结果显示该细胞稳定表达α₁、β₂、γ_2L亚基,构建的α₁β₂γ_2L-GABA_AR-CHO细胞仅在加入四环素(tetracyclin)诱导的情况下表达α₁亚基并与β₂、γ_2L组装成具有功能活性的α₁β₂γ_2L-GABA_AR;对其进行全细胞膜片钳检测研究发现,GABA可对其产生激动效应,引起α₁β₂γ_2L-GABA_AR-CHO细胞产生氯离子通道特征性电流变化,Dia可剂量依赖性地增强GABA对α₁β₂γ_2L-GABA_AR的激动效应;在膜电位检测研究中,获得GABA激动效应EC₅₀为(177.72 ± 15.92)nmol/L,Dia变构效应EC₅₀为(3.63±0.52)μmol/L,拮抗剂Bicuculine拮抗效应IC₅₀为(538.83±29.55)nmol/L。结论: 通过采用诱导表达策略,成功构建了α₁β₂γ_2L-GABA_AR-CHO稳定表达细胞株,该细胞株具有对激动剂、阳性变构剂、拮抗剂特异性检测的药理学功能。     

固相萃取-超高效液相色谱-串联质谱法测定新鲜块菌子实体中α-雄烷醇

本研究建立了一种固相萃取-超高效液相色谱-串联质谱的检测方法,用于检测新鲜块菌子实体中α-雄烷醇(5α-雄甾-16-烯-3α-醇)的含量。新鲜块菌样品经无水乙醇提取,Qasis HLB柱萃取富集后,采用超高效液相色谱-串联质谱进行分析定量。方法学验证结果表明该方法的回收率为88.49%-92.22%;检出限为0.120 9 ng/mL,定量限为0.398 9 ng/mL。该方法简便、精确,适用于新鲜块菌中α-雄烷醇含量的测定。     

盐穗木金属硫蛋白HcMT的体外自由基清除活性及抗氧化能力*

目的: 原核表达盐穗木(Halostachys caspica C. A. Mey.)金属硫蛋白HcMT并探究其抗氧化活性。方法: 构建原核表达载体pET-32a-HcMT,转化至大肠杆菌Escherichia coli BL21,加入Zn²⁺胁迫培养(终浓度为200 μmol/L),分离纯化得到Zn-HcMT,测定Zn-HcMT自由基清除活性和总抗氧化能力,制备复合物Zn-HcMT/TiO₂并做FTIR表征。结果: 通过原核表达获得融合蛋白Zn-HcMT,对·OH、O₂·^-、DPPH自由基具有较强的清除活性,对·OH、O₂·^-的IC₅₀分别为0.386 mg/mL、0.038 mg/mL。融合蛋白浓度为0.01 mg/mL时,对DPPH清除率达(37.43 ± 0.006 8)%,浓度为0.3mg/mL时TEAC(trolox-equivalent antioxidant capacity)值为(1.023 ± 0.01)mmol/L,融合蛋白还原力A₇₀₀为0.142 ± 0.055,FTIR图谱同时表现了Zn-HcMT和TiO₂吸收特性。结论: Zn-HcMT具有良好的清除ROS活性及较强的抗氧化能力,在化妆品领域有潜在应用前景。     

7株野生虫草菌的鉴定及其菌丝体醇提取物对HepG2细胞的抑制活性

结合形态学与ITS序列分析对7株野生虫草真菌进行分类鉴定。MTT法分析它们的菌丝体醇提取物对肝癌HepG2细胞增殖的抑制活性。鉴定结果表明菌株MF7、MF9、MF14为细脚棒束孢Isaria tenuipes,菌株MF11、MF12、MF13为蝉棒束孢Isaria cicadae,菌株MF10为球孢白僵菌Beauveria bassiana;MTT结果显示分离到的3株细脚棒束孢和3株蝉棒束孢的菌丝体醇提取物对HepG2的抑制活性较差,IC₅₀均大于500μg/mL;球孢白僵菌MF10对HepG2细胞有一定抑制作用,IC₅₀值为221.6μg/mL,略强于蝙蝠蛾拟青霉发酵菌丝粉产品金水宝胶囊（IC₅₀=364μg/mL）和中华被毛孢发酵菌丝粉产品百令胶囊（IC₅₀=268.7μg/mL）。另外,发现供对比试验的3株蛹虫草菌株（MF1、MF5、MF15）对HepG2细胞均有较好的抑制作用,其中MF15的发酵菌丝体醇提取物活性最强,IC₅₀为55.56μg/mL,暗示蛹虫草发酵菌丝体具有重要的研究价值。     

长白山阔叶红松林生态系统光能利用率的动态变化及其主控因子

生态系统光能利用率(LUE)反映了植被通过光合作用利用光能吸收和固定大气中CO₂的能力, 是表征生态系统生产力的重要指标。选取长白山温带阔叶红松(Pinus koraiensis)林生态系统为研究对象, 利用涡度相关通量观测数据, 采用直角双曲线方程获取了生态系统光合作用的表观量子效率(ε); 基于总生态系统初级生产力(GEP)与下垫面入射光合有效辐射(Q)的比值得到生态光能利用率(LUE_eco)。研究表明: 在季节尺度上, ε与LUE_eco均表现出显著的单峰变化特征, 并主要受到土壤温度和归一化植被指数(NDVI)的调控, 同时, ε和LUE_eco都受到GEP的显著影响, 而与Q的相关性较弱或无显著相关关系, 但散射辐射的增加在一定程度上有助于提高生态系统的LUE。ε与LUE_eco存在显著的线性正相关关系, 但ε明显高于LUE_eco。2003-2005年, ε与LUE_eco每年最大值的平均值分别为(0.087 ± 0.003)和(0.040 ± 0.002) μmol CO₂·μmol photon^-1, 年际间变异度分别为4.17%和4.25%, 而不同年份之间最大差异均达到8%或8%以上, 从而对模型模拟结果产生明显影响。因此, 在基于光能利用率模型的模拟研究中, 最大LUE的年际变异需要在参数反演和优化中给予重要考虑。     

南方红豆杉内生真菌的抗肿瘤产物震颤毒素的研究

从红豆杉内生真菌青霉菌Penicillum sp. H⑥.1发酵产物中自然析出一种化合物,经图谱检测和文献比对,鉴定为震颤毒素Penitrem A。四唑盐（MTT）比色法研究表明Penitrem A对恶性黑色素瘤细胞A375有一定的抑制作用,且抑制作用随浓度的增大而增强,IC₅₀为27.24μg/mL。本研究是首次从红豆杉内生真菌发酵产物中分离得到震颤毒素Penitrem A,为抗肿瘤药物开发提供了理论参考。     

赭曲霉毒素A和玉米赤霉烯酮-二联胶体金免疫层析试纸条的制备及应用

【背景】真菌毒素为真菌的有毒次级代谢产物,混合污染时毒性显著增强,可对人类和动物健康造成严重伤害。制备二联胶体金免疫层析试纸条,实现对常见真菌毒素混合污染的快速监测,具有重要意义。【目的】制备赭曲霉毒素A (Ochratoxin A,OTA)和玉米赤霉烯酮(Zeralenone,ZEN)金标单克隆抗体,基于免疫层析原理,采用竞争反应模式,建立二联胶体金免疫层析检测法用于污染样品中OTA和ZEN的同时快速检测。【方法】采用柠檬酸钠还原法制备胶体金颗粒,并标记获得两种真菌毒素金标单克隆抗体,通过优化相关条件,建立稳定的二联胶体金免疫层析检测方法,用于同时检测谷物和饲料样品中的OTA和ZEN。【结果】制备的OTA和ZEN二联胶体金试纸条对OTA和ZEN的检测限分别为0.625 ng/mL和1.25 ng/mL,且与谷物和饲料中其它真菌毒素(黄曲霉毒素B1、伏马毒素B1、桔青霉毒素、展青霉毒素和呕吐毒素)均无交叉反应,人工添加试验结果准确。对天然样本检测结果表明该方法与LC-MS/MS一致性良好。【结论】本研究制备的二联胶体金试纸条可用于实际样品中OTA和ZEN的同时快速筛查。     

Development of a liquid chromatography tandem mass spectrometry method for the simultaneous determination of zearalenone,deoxynivalenol and their metabolites in pig serum

A sensitive and selective liquid chromatography tandem mass spectrometry method using negative electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites α-zearalenol, β-zearalenol, zearalanone, α-zearalanol, β-zearalanol and de-epoxy-deoxynivalenol in pig serum. For method development, different sample preparation columns were tested for their suitability for extraction and clean up. Finally, preparation of serum samples was carried out using Oasis? HLB solid-phase extraction (SPE) columns. The analyte concentrations were determined by the use of isotopically labelled internal standards (IS). The method was in-house validated for all analytes. Calibration graphs (0.3–480 ng/ml) were prepared and high degree of linearity was achieved (r?≥?0.99). Results for method precision ranged between 2.7 and 21.5 % for inter-day and between 1.1 and 11.1 % for intra-day. The recoveries were in the range of 82–131 %. Limits of detection and quantification ranged 0.03–0.71 and 0.08–2.37 ng/ml, respectively. The method has been successfully used for quantitative determination of ZEN, DON and their metabolites in pig serum from a feeding trial with practically relevant ZEN and DON concentrations. This method is precise and reproducible and can be used as a multi-biomarker method to assess animal exposure to these mycotoxins and for diagnosis of intoxications.     

Bioherbicidal effect of fumonisin B1, a phytotoxic metabolite naturally produced by Fusarium nygamai, on parasitic weeds of the genus Striga

Fusarium nygamai has been isolated and identified from diseased Striga hermonthica plants collected from sorghum fields in the Sudan. Fumonisin B₁, produced by this fungus, was isolated and purified. In in vitro experiments, the bioherbicidal potential of this mycotoxin was evaluated against S. hermonthica and S. asiatica. Different concentrations (250, 500 and 1000 μg L^-1 of water) of fumonisin B₁, applied during the conditioning phase of the Striga seeds significantly reduced the germination of both species in a range of 19.8-32.2 and 34.5-47.6%, respectively. In addition, the length of the germ tubes was reduced and the germ tubes became brownish. Fumonisin B₁ slightly delayed the emergence of Striga when 250 mg fumonisin B₁ dissolved in 1 L of water were applied into the soil per pot before sowing sorghum. By the way of contrast, fumonisin B₁ was very phytotoxic when Striga seedlings (leaves and stems) of different heights and ages were sprayed with a concentration series of 250, 500 and 1000 μg fumonisin B₁ mL^-1 until run-off, or when plants were injured with a scalpel before fumonisin B₁ was applied into the injury at concentrations of 250 and 500 μg mL^-1. One day after application, the plants showed symptoms of wilting, first at the top and then moving downwards. Leaves turned black and stems desiccated at the point of injuries. Within 4 days after the application, 55% of the plants tested were completely destroyed. In total, at the end of the experiment, 40 days after the treatment, 85% of the treated plants were killed by fumonisin B_1, regardless of the concentration applied and the application technique used.     

基于超声清洗的树木叶面颗粒物粒径分布与吸滞效率研究——以银杏和油松为例

明确在常规叶片清洗方法(泡洗或泡洗+刷洗)上增加超声清洗对叶面各径级颗粒物滞纳量定量评估的影响, 并在此基础上研究叶面颗粒物的粒径分布和吸滞效率, 可进一步提高城市树木大气颗粒物吸滞能力的定量评估精度。该文以城市森林建设常用阔叶树种银杏(Ginkgo biloba)和针叶树种油松(Pinus tabuliformis)为研究对象, 于雨后(降水量>15 mm) 4天(短滞尘时长)和14天(长滞尘时长)分别采集叶样, 并依次对其进行泡洗(WC)、刷洗(BC)、超声清洗(UC)等洗脱程序, 然后对每个清洗步骤下叶片洗脱液中颗粒物的质量和粒径分布进行测试, 并依此估算叶片各径级颗粒物的吸滞效率。结果表明, 以“泡洗+刷洗+超声清洗”清洗流程的测试结果为参照, 若只对叶片进行泡洗, 则银杏和油松对大气颗粒物(PM₁, 粒径d ≤1 µm)、PM_2.5 (d ≤ 2.5 µm)、PM₅ (d ≤ 5 µm)、PM₁₀ (d ≤ 10 µm)吸滞量会分别被低估约一半(54%、53%、53%和53%)和40% (42%、42%、42%和42%); 若只进行“泡洗+刷洗”, 则银杏和油松对相应径级颗粒物的吸滞量仍会分别被低估约15% (17%、16%、15%和15%)和20% (21%、20%、20%和20%)。油松叶面颗粒物粒径分布呈双峰曲线, 而银杏叶面颗粒物粒径则呈单峰分布, 且银杏叶面颗粒物平均粒径在短、长滞尘时长下均大于油松。油松叶片对PM₁、PM_2.5、PM₅、PM₁₀和总悬浮颗粒物的吸滞效率分别为8.96、23.92、23.96、23.96和23.96 mg·m^-2·d^-1, 分别比银杏叶片高112%、73%、34%、37%和42%。     

A rapid method for simultaneous determination of zearalenone, α-zearalenol, β-zearalenol, zearalanone, α-zearalanol and β-zearalanol in traditional Chinese medicines by ultra-high-performance liquid chromatography-tandem mass spectrometry

A rapid and reliable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of zearalenone (ZEN), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL) in traditional Chinese medicines (TCMs) was developed. The development of the method and investigations of the matrix influence were described in particular. After evaluation of the matrix effects of different TCMs, i.e., rhizomes and roots, seeds, flowers, grasses and leaves, by the post-extraction spiked method, a reliable sample clean-up method based on home-made clean-up cartridges, a suitable internal standard and the matrix calibration were combined using to minimize the matrix effects to ensure the accuracy of the method. The established method was further validated by determining the linearity (R(2)≥0.9990), sensitivity (limit of quantitation 0.11-0.99 ng mL(-1)), average recovery (86.6-113.5%) and precision (relative standard deviation ≤13.5%). It was shown to be a suitable method for simultaneous determination of ZEN, α-ZOL, β-ZOL, ZAN, α-ZAL and β-ZAL in different TCMs. Finally, the established method was successfully applied to the determination of the six mycotoxins in various TCMs and the results were presented to provide relevant insights to researchers in TCM analysis.     

Dynamics and regulations of ecosystem light use efficiency in a broad-leaved Korean pine mixed forest,Changbai Mountain

Aims Ecosystem light use efficiency (LUE) reflects the ability of CO₂ uptake and light utilization via photosynthesis, which is a key parameter in ecosystem models to evaluate ecosystem productivity. The objectives of this study were to: (1) compare the differences of LUE derived from different methods; (2) elucidate the seasonal dynamics of LUE and its regulatory factors; and (3) evaluate the maximum LUE (LUE_max) and its variability based on eddy-covariance (EC) flux.Methods Using the flux data from an EC tower during 2003-2005 at a broad-leaved Korean pine (Pinus koraiensis) mixed forest, Changbai Mountain, two types of LUE indicators were generated from: 1) the apparent quantum yield (ε) estimated with rectangular hyperbolic curve, and 2) the ecological light use efficiency (LUE_eco) calculated as the ratio between gross ecosystem productivity (GEP) and photosynthetically-active radiation (Q).Important findings The seasonal variation of ε and LUE_eco appeared a unimodal pattern within a year, with the variations significantly dominated by soil surface temperature and Normalized Difference Vegetation Index (NDVI). A positive correlation between GEP and LUE was found for both ε and LUE_eco, with the effect of Q on LUE relatively weak. The increase in diffusion radiation appeared favorable for enhanced LUE. Generally, there was a significant positive relationship between ε and LUE_eco, while ε was higher than LUE_eco, especially during the mid-season. The annual maximum value of ε and LUE_eco was (0.087 ± 0.003) and (0.040 ± 0.002) μmol CO₂·μmol photon^-1 over the three years, respectively. The interannual variability of LUE_max for ε and LUE_eco was 4.17% and 4.25%, respectively, with a maximum difference of >8%, likely resulted from considerable uncertainty in model simulations. Our results indicated that the inversion and optimization of maximum LUE should be taken seriously in the application of LUE models.