An assay of ganglioside using fluorescence image analysis on a thin-layer chromatography plate

A new and simple fluorometric method for determine quantities of gangliosides ranging from pico- to nanomoles is reported. Spraying hydrochloric acid followed by a heating treatment, sugars (glucose, galactose, fucose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid (sialic acid)), gangliosides (G(M1), G(D1a), G(T1b)), and asialoganglioside (asialoG(M1)) on thin-layer chromatography plates produced fluorescence under 365-nm UV light. This fluorescence production of each sample was greatly dependent on the heating temperature. As sialic acid fluoresced readily at lower temperature (approximately 90 degrees C), we were able to distinguish sialic acid easily from other sugars tested. To determine gangliosides based on this sialic acid fluorescence, calibration curves for gangliosides were obtained by high-performance thin-layer chromatography and the image-analyzing system equipped with a CCD camera. The observed fluorescence images were analyzed using image-analyzing software packages and the determined calibration curves for ganglioside-bound sialic acids were reproducible and showed a high linearity in a wide range from 47 pmol to 4.5 nmol. Since the fluorescence from sialic acid is easily measurable on TLC plates and is sensitive over a wide range of sample concentration, the present method is applicable for quantitative determination of gangliosides.     

采用荧光光谱法研究DDAF与BSA的相互作用

研究新型反离子季铵盐DDAF与BSA的相互作用。通过单因素法,选出适宜的作用时间、Na+浓度和pH值;并采用荧光光谱法和三维荧光光谱法研究DDAF与BSA的相互作用机制。作用时间15 min、钠离子浓度为0.04 mol/L、pH值为待测液的初始pH值为适宜的实验条件;不同温度(290.15,296.15,303.15,310.15 K)下二者的双分子猝灭常数(Kq)分别为7.03×10~(10),8.53×10~(10),1.09×10~(11),1.12×10~(11) L/(mol·s);结合位点数(n)分别为1.17、0.84、0.97和1.02;?H和?S均大于0;三维荧光光谱显示,加入DDAF后,BSA的荧光峰发生蓝移,且荧光强度下降16.63%。DDAF和BSA为混合猝灭;二者以1∶1结合;二者间的作用力是疏水作用力;DDAF使BSA氨基酸残基周围的疏水环境增强。     

Surface plasmon field-enhanced fluorescence spectroscopy studies of the interaction between an antibody and its surface-coupled antigen

Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) uses the greatly enhanced electromagnetic field of a surface plasmon mode for the excitation of surface-confined fluorophores. The ability to simultaneously monitor the interfacial refractive index changes and the fluorescence signals in real time offers a huge potential for applications of SPFS in surface immunoreaction detection. In this study, gold surfaces were functionalized by mixed self-assembled monolayers exposing an antigen (biotin) at a density that was varied over a wide range. Specific antibody-antigen interactions were observed for anti-biotin antibody solutions passing over the surfaces with a rather high flow speed driven by a home-built liquid-handling system. First, the use of the fluorophores Cy5 and Alexa Fluor 647 in SFPS-based immunoassays was investigated. It was found that Cy5 exhibits strong self-quenching, which makes it rather unsuitable for quantitative measurements. For the in situ measurement of the binding kinetics, an angular "detuning" effect was confirmed to negatively interfere with the fluorescence signal in cases where large SPR signals were detected. An in-depth comparison between the SPR and the fluorescence signal reveals that the fluorescence yield of the dyes depends strongly on the separation distance from the gold surface. And finally, we stress the ability of SPFS to detect binding to surfaces containing extremely diluted antigen density, where the SPR signal failed to follow.     

荧光光谱法研究头孢孟多脂与牛血清白蛋白的相互作用

模拟生理条件下,用荧光光谱法和紫外-可见吸收光谱法研究头孢孟多酯和牛血清白蛋白(BSA)结合反应的特征。研究表明:头孢孟多酯与BSA形成复合物,从而猝灭BSA的内源性荧光,该过程为静态猝灭过程。根据SternVolmer方程得出不同温度下结合位点数n和结合常数Ka;结合位点位于BSA的亚结构IIA中。通过计算相应的热力学参数,确定头孢孟多酯与BSA之间的作用力主要为静电作用力。利用同步荧光光谱探讨了头孢孟多酯与BSA作用前后白蛋白的构型变化。Hill系数nH1,表明头孢孟多酯有弱的负协同作用。此研究不仅对于揭示体内药物动力学问题和指导临床合理用药具有一定意义,而且对药物分子设计及新药开发等也具有重要指导意义。     

Analysis of n-alkanes in water samples by means of headspace solvent microextraction and gas chromatography

A simple and efficient headspace solvent microextraction (HSME) was developed for the simultaneous determination of the trace concentrations of some n-alkanes in water samples. Therefore, a microdrop of an organic solvent was extruded from the needle tip of a gas chromatographic syringe to the headspace above the surface of the solution in a sealed vial. Then the volatile organic compounds are extracted and concentrated in the microdrop. Next, the microdrop was retracted into the microsyringe and injected directly into the gas chromatograph. Experimental parameters which control the performance of HSME such as the type of microextraction solvent, organic drop and sample volume, sample stirring rate, sample solution and microsyringe needle temperatures, salt addition and exposure time profiles were investigated and optimized. Finally, the enrichment factor, dynamic linear range (DLR), limit of detection (LOD) and precision of the method were evaluated. Using optimum extraction conditions, good linearity with correlation coefficients in the range of 0.995相似文献   

船舶螺旋桨叶片与艉部湍流场互作用噪声的预报研究

低噪声特性是螺旋桨设计者所追求的目标之一。当船尾的螺旋桨周期性旋转在空间和瞬时随机的非均匀流场中时,在螺旋桨叶片上就会产生升力脉动,从而辐射出离散谱噪声和低频宽带噪声。利用湍流谱和FfowcsWilliams-Hawkings方程可以获得低频宽带噪声级。这种方法可用于实船螺旋桨噪声预报和低噪声设计,这对提高舰船安静化程度有重要的现实意义。     

荧光光谱与Zeta电位法研究十二烷基二甲基氧化胺与DNA的相互作用

通过荧光光谱与Zeta电位法研究了十二烷基二甲基氧化胺(DDAO)与鲑鱼精DNA的相互作用.非离子态DDAO胶束与DNA间存在弱的疏水相互作用.阳离子态DDAO与DNA的强烈静电相互作用导致体系荧光猝灭.胶束的形成有利于DNA构象的压缩与体系Zeta电位的提高.外加NaCl的静电屏蔽作用减弱了DDAO胶束与DNA间的静电引力.     

三维荧光光谱法和圆二色谱法研究氨基比林与牛血清白蛋白分子的相互作用

该文在模拟生理条件下,采用荧光光谱法、三维荧光光谱法以及圆二色谱法,研究氨基比林(PYM)与牛血清白蛋白(BSA)之间的相互作用。研究结果表明PYM对BSA有强烈的荧光猝灭作用,其荧光猝灭机制为静态猝灭。由热力学参数判定PYM和BSA的主要作用力为氢键和范德华力,并且相互作用是自发进行的。根据F觟rster的偶极-偶极非辐射能量转移理论可以得出PYM与BSA之间的结合距离为2.09 nm。利用三维荧光光谱和圆二色谱技术,分析PYM对BSA蛋白构象的影响,表明PYM与BSA相互作用后使BSA的微环境和构象发生改变。     

用气相色谱法对水中有机磷农药的测定

本文采用固相萃取法预处理,然后用带有火焰光度检测器(FPD)的气相色谱仪测定有机磷农药。结果表明,该法具有简单、快速、准确的持点。     

Anaerobic biodegradation of an organic by-products leachate by interaction with different mine tailings

Deinking by-products from paper recycling have been used as cover materials on acid-producing tailings. Due to residual cellulose, anaerobic degradation leads to the production of an organic-rich leachate, which may percolate through the tailings. This study aims at describing the influence of the tailings’ characteristics on the attenuation of organic matter in leachate and at discussing the degradation mechanisms. To this end, leachate was mixed with different types of tailings, including three unoxidized tailings with varying acid generation potentials and one tailing in three states of oxidation. Regularly, selected biochemical parameters were analyzed to access the evolution of organic components. The results show that when leachate from deinking by-products was placed in contact with tailings, phase and acid–base balance reactions took place in the beginning. Subsequently, oxidation–reduction reactions dominated the chemistry of the system. The type, the constituents, and the state of oxidation of the mine tailings condition the mechanisms of biodegradation of organic components. Methanogenesis was predominant in the control sample (pure organic leachate) but was absent in all leachate-tailings mixtures. No biodegradation was observed in the liquid phase for oxidized tailings and the organic concentration remained constant for unoxidized tailings, independently of the acid-generation potential. The biodegradation efficiency was optimal when the leachate was in contact with a mixture of oxidized and unoxidized tailings due to sulfate-reduction.     

Direct microextraction and analysis of rough-type lipopolysaccharides by combined thin-layer chromatography and MALDI mass spectrometry

A rapid method for the microscale extraction of lipopolysaccharides (endotoxins, LPSs) from rough-type Gram-negative bacteria was developed using thin-layer chromatography (TLC) combined with improved conditions for LPS analysis by mass spectrometry. TLC of intact bacteria on silica gel plates in an appropriate solvent selectively extracted and separated their LPS components. The bands of molecular species were scraped from the plates after nondestructive visualization, directly mixed with matrix, and analyzed by laser desorption time-of-flight mass spectrometry. Lipids A and Re-type LPSs were analyzed after transfer to a membrane. Adding citric acid to the matrix gave greatly improved mass spectra. The system allows characterization of bacterial LPS at the microscale level and is equally well applicable to heterogeneous LPS and lipid A preparations (Escherichia coli lipid A and Bordetella lipopolysaccharides were used). The technique provides a rapid determination of the heterogeneity of unmodified preparations and the determination of the molecular weight of each separated component.     

Real-time image acquisition for absorbance detection and quantification in thin-layer chromatography

This paper presents the first quantitative study of real-time acquisition of images of spots on thin-layer chromatographic plates during development. Procedures are described for imaging using a CCD camera and for image processing, incorporating corrections for fixed pattern effects and compensation for the moving solvent front, to measure the absorbance of the analyte. Imaging of Sudan II was carried out in transmission mode, and peak areas were found to be time-independent. Quantification of the relationship between peak area and sample loading was established over the range 1-50 ng. After averaging 55 images obtained during a single chromatographic run, which attenuates noise contributions from local nonuniformities in the sorbent layer, precision and detection limits were found to be comparable with values obtained in previous work using offline measurements.