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首页> 外文期刊>The Analyst: The Analytical Journal of the Royal Society of Chemistry: A Monthly International Publication Dealing with All Branches of Analytical Chemistry >Microchip electrophoresis utilizing an in situ photopolymerized Phos-tag binding polyacrylamide gel for specific entrapment and analysis of phosphorylated compounds
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Microchip electrophoresis utilizing an in situ photopolymerized Phos-tag binding polyacrylamide gel for specific entrapment and analysis of phosphorylated compounds

机译：微芯片电泳利用原位光聚合的Phos标签结合聚丙烯酰胺凝胶，用于特异性涂覆和分析磷酸化合物

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摘要

A method was developed for the specific entrapment and separation of phosphorylated compounds using a Phos-tag polyacrylamide gel fabricated at the channel crossing point of a microfluidic electrophoresis chip. The channel intersection of the poly(methyl methacrylate)-made microchip was filled with a solution comprising acrylamide, N, N-methylene-bis-acrylamide, Phos-tag acrylamide, and 2,2'-azobis [2-methyl-N-(2-hydroxyethyl) propionamide], which functioned as a photocatalytic initiator. In situ polymerization at the channel crossing point was performed by irradiation with a UV LED laser beam. The fabricated Phos-tag gel (100 x 100 x 30 mu m) contains ca. 20 fmol of the Phos-tag group and therefore could entrap phosphorylated compounds at the femtomolar level. The electrophoretically trapped phosphorylated compounds were released from the gel by switching the voltage to deliver high concentrations of phosphate and EDTA in a background electrolyte. The broad sample band eluted from the gel was effectively reconcentrated at the boundary of a pH junction generated by sodium ions delivered from the outlet reservoir. The reconcentrated sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, the phosphorylated compounds were concentrated by a factor of 100-fold, and the peak resolution was comparable to that obtained by pinched injection. This method was successfully utilized to preconcentrate and analyze phosphorylated peptides in a complex peptide mixture.
机译：使用在微流体电泳芯片的通道交叉点制造在微流体电泳芯片的通道交叉点的Phos-Tag聚丙烯酰胺凝胶来开发一种方法，用于特异性截留和分离磷酸化化合物。聚（甲基丙烯酸甲酯）-MADE微芯片的沟道交叉点填充了包含丙烯酰胺，N，N-亚甲基 - 双丙烯酰胺，丙烯酰胺和2,2'-偶氮基的溶液[2-甲基-N- （2-羟乙基）丙酰胺]，其用作光催化引发剂。通过用UV LED激光束照射进行通道交叉点处的原位聚合。制造的PHOS标签凝胶（100×100×30μm）含有CA. 20个Fmol的Phos标签组，因此可以在Femtomolar水平上捕获磷酸化化合物。通过切换电压从凝胶中释放电泳被捕获的磷酸化化合物，以在背景电解质中输送高浓度的磷酸盐和EDTA。从凝胶中洗脱的宽样品带在由从出口贮存器输送的钠离子产生的pH结的边界处有效地重新分解。然后在分离通道的末端分离和荧光检测重新浓缩的样品组分并荧光地检测。在优化的条件下，将磷酸化化合物浓缩100倍，峰值分辨率与通过夹持注射获得的峰值分辨率相当。该方法已成功地利用以预浓缩，并在复杂的肽混合物中分析磷酸化肽。

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