Effect of monosaccharide composition, glycosidic linkage position and anomericity on the electrophoretic mobility of labeled oligosaccharides

Fluorophore-assisted carbohydrate electrophoresis (FACE) is useful for separation and characterization of oligosaccharides from various sources and for comparing several samples at once. While characterizing fungal surface glycans by FACE we observed that samples and standards of the same mass did not comigrate as expected. Subsequent experiments showed that the samples did not contain contaminating sugars. Therefore, our observation suggested that glycan electrophoretic mobility is affected by factors in addition to molecular mass. This work assesses the contribution of monosaccharide composition, linkage position, and linkage anomericity to glycan mobility. Commercially available (and synthesized when available) bioses of known composition were derivatized with a charged fluorophore, and electrophoretic mobilities compared in a slab gel format. The results indicate that all three parameters mentioned above affect observed migration. Further, no migration patterns emerged to suggest a set of rules for assigning band identity based on mobility alone. These results emphasize the importance of including known, matched, standards to facilitate interpretation of FACE data.     

Microscale analysis of mucin-type O-glycans by a coordinated fluorophore-assisted carbohydrate electrophoresis and mass spectrometry approach

The total glycan moiety was released in a single step from native glycoproteins by a nonreductive beta-elimination procedure. The generated oligosaccharides were further derivatized either with the hydrophobic fluorophore 2-aminoacridone (AMAC) or the charged 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) fluorophore, and the resulting fluorescent derivatives were separated according to their hydrodynamic size or charge with high-resolution gel electrophoresis. Both N- and O-glycans released by this beta-elimination procedure might be analyzed simultaneously. AMAC derivatization allows a rapid separation of neutral and charged oligosaccharides without prior fractionation. Derivatized oligosaccharide species were then eluted from the gel slices and analyzed by mass spectrometry. This methodology allowed the rapid structural characterization of each glycan in term of monosaccharide composition and sequence. Using this technique we were able to screen several heterogeneous O-glycan mixtures isolated at the picomolar range from reference glycoproteins or mucins.     

Boronate affinity saccharide electrophoresis: a novel carbohydrate analysis tool

The incorporation of specialised carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for fluorophore-assisted carbohydrate electrophoresis greatly improved the effective separation of saccharides that show similar mobilities in standard electrophoresis. Polyacrylamide gel electrophoresis using methacrylamido phenylboronic acid in low loading (typically 0.5-1% dry weight) was unequivocally shown to alter retention of labelled saccharides depending on their boronate affinity. While conventional fluorophore-assisted carbohydrate electrophoresis of 2-aminoacridone labelled glucose oligomers showed an inverted parabolic migration, an undesired trait of small oligosaccharides labelled with this neutral fluorophore, boron affinity saccharide electrophoresis separation of these carbohydrates completely restored their predicted running order, based on their charge/mass ratio, and resulted in improved separation of the analyte saccharides. These results exemplify boron affinity saccharide electrophoresis as an important new technique for analysing carbohydrates and sugar-containing molecules.     

基于氧化石墨烯荧光适体传感器的胰岛素检测

采用荧光基团(FAM)标记的核酸适体作为识别元件,氧化石墨烯为淬灭剂,建立了一种高选择性、高灵敏度的核酸适体传感器.核酸适体与氧化石墨烯结合后,荧光淬灭,此时溶液无荧光;加入胰岛素后,溶液中荧光得到恢复.利用荧光分析法检测加入胰岛素前后,溶液中荧光强度的变化,获取了荧光适体传感器的线性度和灵敏度,实现对胰岛素浓度的测定.结果表明,在5×10-8 ～ 1×10-5 mol/L范围内,胰岛素的浓度与溶液中荧光强度有良好的线性关系,检出限为10 nmol/L.采用此方法检测胰岛素,操作简便,检测速度快,准确性高,选择性好,检出限低.     

Long‐Term Live‐Cell STED Nanoscopy of Primary and Cultured Cells with the Plasma Membrane HIDE Probe DiI‐SiR

Super‐resolution imaging of live cells over extended time periods with high temporal resolution requires high‐density labeling and extraordinary fluorophore photostability. Herein, we achieve this goal by combining the attributes of the high‐density plasma membrane probe DiI‐TCO and the photostable STED dye SiR‐Tz. These components undergo rapid tetrazine ligation within the plasma membrane to generate the HIDE probe DiI‐SiR. Using DiI‐SiR, we visualized filopodia dynamics in HeLa cells over 25 min at 0.5 s temporal resolution, and visualized dynamic contact‐mediated repulsion events in primary mouse hippocampal neurons over 9 min at 2 s temporal resolution. HIDE probes such as DiI‐SiR are non‐toxic and do not require transfection, and their apparent photostability significantly improves the ability to monitor dynamic processes in live cells at super‐resolution over biologically relevant timescales.     

Azo-pyrene–based fluorescent sensor of reductive cleavage of isomeric azo functional group

In this study we investigated the reductive azo cleavage of an azo compound presenting a pyrene fluorophore (Azo-py). Because of dramatic changes in its fluorescence, Azo-py could be used as a monitoring system for the reductive azo cleavage. Electron transfer from the pyrene unit to the azo moiety induced fluorescence quenching; this quenched fluorescence was recovered after the reductive azo cleavage. IR and NMR spectroscopy were used to study the various structural states. The rate of reductive cleavage of the azo compound, determined through fluorescence monitoring, depended on its structural state: the cleavage of trans-Azo-py was much faster than that of the cis-Azo-py. Furthermore, the Azo-py fluorophore was highly sensitive to the presence of zinc, but not other metal compounds, and the pH.     

Naphthalene derivatives of a conformationally locked GFP chromophore with large stokes shifts

The active development of fluorescence microscopy requires an increase in the variety of the dyes and their characteristics. Compounds with a large Stokes shift, i.e., a large difference between the positions of the absorption and emission maxima, have attracted the interest of researchers as a tool that can be used for multicolor labeling. One of the known approaches to increase the Stokes shift is the introduction of additional polycyclic fragments. Herein, we report novel derivatives of a conformationally locked GFP chromophore containing the naphthalene ring. The proposed modification leads not only to the enhancement of the Stokes shift up to 100 nm but also leads to the noticeable red-shift of the emission and absorption spectra in contrast with the corresponding derivatives with one benzene ring.     

Structure-fluorescence relationship of push-pull 2-phenylbenzothiazole derivatives designed based on the firefly light-emitter

6-Dimethylamino-2-phenylbenzothiazole (1a) mimicking the firefly oxyluciferin structure and the derivatives with an electron-withdrawing substituent on the phenyl group were prepared, and their fluorescence properties were investigated in various solvents. 1a showed solvatochromic fluorescence with good fluorescence quantum yields (Φ_f >0.8). The introduction of an electron-withdrawing group led to a red-shift of the emission maximum. In particular, the derivatives with the 2,2-dicyanoethenyl and (1,3-dihydro-1,3-dioxo-2H-inden-2-ylidene)methyl groups showed near-infrared fluorescence in chloroform. In addition, the derivative with the phenylimine moiety showed efficient solid-state fluorescence, resulted from a molecular arrangement inhibiting intermolecular interactions for quenching the fluorescence state in crystals.