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免疫毛细管电泳-激光诱导荧光分析DNA加合物的方法学研究 总被引:2,自引:1,他引:1
DNA加合物是一类重要的生物标志物,可应用于人体致癌物暴露监测、癌症风险评价和人群易感性研究。DNA加合物作为生物标志物的应用需要安全、灵敏、快速的先进分析技术。我们利用免疫毛细管电泳-激光诱导荧光分析,发展了高灵敏的DNA加合物分析方法和技术。本文主要介绍了相关的仪器研制及方法学研究。方法学研究涉及DNA加合物荧光探针的合成和表征、抗体与DNA加合物的相互作用及其结合计量学、抗原-抗体复合物的稳定化和DNA驱动电泳聚焦技术。 相似文献
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以羟丙甲基纤维素和非交联聚丙烯酰胺溶液为筛分介质,将毛细管电泳-激光诱导荧光法用于DNA片段及基因扩增产物的分离检测。探讨了非胶筛分介质中高分子化合物的浓度、电解质的浓度、内插试剂用量等对DNA片段分析检测的影响;考察了DNA片段迁移时间和峰面积的重现性及DNA片段定量检测的关系,建立了一种快速、灵敏的DNA片段及基因扩增物分离检测方法。 相似文献
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通过理论推导和实验验证表明;适当稀释DNA样品溶液,采用流体力学进样或电动进样都不会较大地减低峰高,而DNA片段毛细管电泳的分离效率和分离度还能有所提高。采用稀释样品的方法可提高DNA样品的使用效率。采用羟乙基纤维素无胶筛分介质分离了DNA片段。用激光诱导荧光(氩离子激光器,488nm)电荷耦合器件检测。用低浓度的筛分介质(0.4%)分离了分子质量较大的ADNA-HindⅢ全部8个片段(12bp~23130bP)。用高浓度的筛分介质(1.6%)分离分子质量较小的pBR322-HaeⅢ22个片段(18bp~587bp)。 相似文献
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以羟丙甲基纤维素和非交联聚丙烯酰胺浴液为筛分介质,将毛细管电泳-激光诱导荧光法用于DNA片段及基因扩增产物的分离检测。探讨了非胶筛分介质中高分子化合物的浓度、电解质的浓度、内插试剂用量等对DNA片段分离检测的影响;考察了DNA片段迁移时间和峰面积的重现性及DNA片段定量检测的关系。建立了一种快速、灵敏的DNA片段及基因扩增产物分离检测方法。 相似文献
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增强型电荷耦合器件用于毛细管电泳激光诱导荧光的检测 总被引:3,自引:0,他引:3
增强型电荷耦合器件用于毛细管电泳激光诱导荧光的检测马明生,吴晓军,刘国诠(中国科学院化学研究所,北京,100080)关键词增强型电荷耦合器件,激光诱导荧光,毛细管电泳,异硫氰酸荧光黄近年来,毛细管电泳技术在生化分离、分析中得到广泛的应用。目前,毛细管... 相似文献
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利用激光喇曼装置进行毛细管电泳—激光诱导荧光实验 总被引:4,自引:0,他引:4
细管电泳(electrophoresis,CE)是一种高效、快速的新型分析方法,尤其适合于肽、蛋白质及核苷酸等生理活性大分子的分离。CE通常以内径小于100μm弹性石英管为分离通道,受光程和进样量的限制,常用的紫外吸收检测无法满足低浓度物质检测的需求... 相似文献
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毛细管电泳微流控芯片分离-激光诱导荧光(LIF)检测DNA片段是近年来微流控分析系统中研究得较为成功的领域,该方向的研究成果极大地促进了微流控分析系统的发展.在相关的报道中,待分析样品和系统运行溶液仍然主要使用手工操作. 相似文献
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《Analytical letters》2012,45(11):1944-1963
Abstract This is the first attempt for the direct detection of polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human placental DNA samples by solid-matrix phosphorescence (SMP). Six samples were investigated, and SMP emission spectra and the corresponding second derivative SMP spectra were obtained for all the samples. Numerous excitation and emission wavelengths were studied for detecting PAH-DNA adducts. Second derivative SMP spectra indicated the presence of PAH-DNA adducts, whereas the longer SMP emission region proved fruitful for detecting adducts in the placental DNA samples. The SMP results for the samples strongly implied that a variety of PAH-DNA adducts could be present. 相似文献
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通常用于DNA定量的主要方法有聚合物酶链反应(PCR)、紫外光谱法和荧光光谱法等.PCR测定DNA的灵敏度较高,但操作繁琐费时,而且对序列依赖性较强[1].紫外吸收光谱法可直接用于DNA的测定,但它的灵敏度较低且较容易受到DNA样品中的单链核苷酸和其它杂质的干扰.荧光光谱法不但有较高的灵敏度,而且对DNA的选择性也较好.目前已建立了许多荧光测定DNA的方法,这些测定方法一般都是基于DNA对荧光探针的荧光增强的原理而实现的.通常采用荧光光谱仪、荧光测读分析仪、流式细胞仪等测定荧光标记的DNA的荧光信号,但这些仪器均存在样品消耗量较大、仪器价格昂贵、分析时间较长、不易自动化等缺陷.毛细管电泳仪已经成为许多分析实验室中常用的DNA分离检测工具,将其应用于DNA的定量检测有望克服上述仪器的一些缺陷.Landers等以YO-PRO-1和PicoGreen为DNA标记试剂, 在毛细管电泳仪上对DNA含量进行了较好的分析[2]. 相似文献
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Sung Hee Ahn Kyung Man Park Jeong Hee Moon Seong Hoon Lee Myung Soo Kim 《Journal of the American Society for Mass Spectrometry》2016,27(11):1887-1890
The utility of sodium ion adducts produced by matrix-assisted laser desorption ionization for the quantification of analytes with multiple oxygen atoms was evaluated. Uses of homogeneous solid samples and temperature control allowed the acquisition of reproducible spectra. The method resulted in a direct proportionality between the ion abundance ratio I([A?+?Na]+)/I([M?+?Na]+) and the analyte concentration, which could be used as a calibration curve. This was demonstrated for carbohydrates, glycans, and polyether diols with dynamic range exceeding three orders of magnitude. 相似文献
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本文建立了一种同时检测DNA样品中Ethylidene-dG和Propano-dG含量的液相色谱-串联质谱(LC-MS/MS)方法,方法的精密度较好(RSD6%),检出限分别为0.010ng/mL和0.005ng/mL,回收率在97.2%~101.6%之间。同时,以体外小牛胸腺DNA为模型,选取不同乙醛暴露剂量(0、0.001、0.01、0.05、0.1、0.5和1.0mmol/L)和不同的暴露时间(0、2、4、10、12、20和24h),结果显示在体外Propano-dG加合物的生成需有氨基酸作为催化剂,且小牛胸腺DNA中乙醛-DNA加合物的含量随着染毒剂量和染毒时间的增加而升高,存在剂量-效应和时间-效应关系。 相似文献
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A novel method for the determination of proteins at nanogram levels was proposed based on the decrease of resonance light
scattering (RLS) signal resulting from the interaction of dibromo-o-nitrophenylfluorone (DBONPF)-sodium lauroyl glutamate
(SLG) with proteins. At pH 2.97, the decrease RLS intensity was proportional to the concentration of proteins in the range
of nanogram levels with 3σ detection limits being 3.4 ng mL−1 for bovine serum albumin (BSA), 1.7 ng mL−1 for human serum albumin (HSA), 4.1 ng mL−1 for γ-globulin (γ-IgG), 4.4 ng mL−1 for egg albumin, 6.2 ng mL−1 for pepsin (Pep) and 3.7 ng mL−1 for α-chymotrypsin (Chy). The method is no protein-to-protein variability, simple, rapid, practical and relatively free
from interference from coexisting substance, as well as much more sensitive than most of the reported methods. The proposed
method was successfully applied to determine total protein in human serum samples. 相似文献
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Quantification of DNA in a forensic sample is of major importance for proper DNA amplification and STR profiling. Several methods have been developed to quantify DNA, from basic UV spectrometry, through gel-based techniques, to dye staining, blotting techniques, and, very recently, DNA amplification methods (polymerase chain reaction, PCR). Early techniques simply measured total DNA, but newer techniques can specifically measure human DNA while excluding non-human DNA (foodstuff, animal, or bacterial contamination). These newer assays can be faster and less expensive than traditional methods, making them ideal for the busy forensic laboratory. This paper reviews classic and newer quantification techniques and presents methods recently developed by the authors on the basis of PCR of Alu sequences. 相似文献
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Hiroki Yamamoto Takuya Fujiwara Takashi Funatsu Makoto Tsunoda 《Molecules (Basel, Switzerland)》2021,26(8)
Biothiols, such as cysteine and glutathione, play important roles in various intracellular reactions represented by the redox equilibrium against oxidative stress. In this study, a method for intracellular thiol quantification using HPLC-fluorescence detection was developed. Thiols were derivatized with a thiol-specific fluorescence derivatization reagent, viz. ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), followed by reversed-phase separation on an InertSustain AQ-C18 column. Six different SBD-thiols (homocysteine, cysteine, cysteinylglycine, γ-glutamylcysteine, glutathione, and N-acetylcysteine as an internal standard) were separated within 30 min using a citric buffer (pH 3.0)/MeOH mobile phase. The calibration curves of all the SBD-thiols had strong linearity (R2 > 0.999). Using this developed method, the thiol concentrations of human chronic myelogenous leukemia K562 cell samples were found to be 5.5–153 pmol/1 × 106 cells. The time-dependent effect of a thiol scavenger, viz. N-ethyl maleimide, on intracellular thiol concentrations was also quantified. This method is useful for elucidating the role of intracellular sulfur metabolism. 相似文献