Llopis Shawn D, Stryjewski Wieslaw, Soper Steven A
Louisiana State University, Department of Chemistry, Baton Rouge, LA 70803-1804, USA.
Electrophoresis. 2004 Nov;25(21-22):3810-9. doi: 10.1002/elps.200406054.
High aspect-ratio microstructures were hot-embossed in polymer substrates with a molding tool fabricated using lithography/electroplating/forming (LIGA). The resulting devices were used for the electrophoretic separation of oligonucleotides labeled with near-infrared (near-IR) dyes. Near-IR time-resolved fluorescence was used as an identification method for the labeling dyes. The detection apparatus consisted of a pulsed laser diode operating at 680 nm, a single-photon avalanche diode, an integrated microscope, and a PC-board incorporating time-correlated single photon counting electronics. Investigation of the optical quality and amount of autofluorescence generated from different polymer substrates was carried out in the near-IR region for determining compatibility with time-resolved fluorescence. Our results revealed that of several poly(methylmethacrylate)(PMMA) substrates, brand Plexiglas offered minimal replication errors in the embossed features using appropriate embossing conditions with low background fluorescence contributions to the observed decay. Near-IR dye-labeled oligonucleotides were separated to determine the applicability of fluorescence lifetime discrimination between Cy5.5 (tauf = 930 ps) and IRD700 (tauf = 851 ps) labeling dyes during the microchip separation. These dyes were used to label T-fragments (thymine) of an M13mp18 ssDNA template. The DNA ladders were electrophoresed at 130 V/cm in a 4% linear polyacrylamide gel (LPA) gel matrix in a 9.5 cm long serpentine channel heated to 50 degrees C. The electropherogram revealed that the lifetimes could be accurately read well beyond 450 bases, although single-base pair resolution in the electropherogram was difficult to achieve due to potential solute-wall interactions in the polymer microdevice or the electroosmotic flow (EOF) properties of the device. The relative standard deviations secured for individual bands in the electropherogram were similar to those obtained using capillary gel electrophoresis, in spite of the lower load volume.
使用光刻/电镀/成型（LIGA）制造的成型工具在聚合物基板上热压印出高纵横比的微结构。所得器件用于对用近红外（near-IR）染料标记的寡核苷酸进行电泳分离。近红外时间分辨荧光被用作标记染料的识别方法。检测装置由一个工作在680nm的脉冲激光二极管、一个单光子雪崩二极管、一台集成显微镜以及一块包含时间相关单光子计数电子设备的印刷电路板组成。在近红外区域对不同聚合物基板产生的光学质量和自发荧光量进行了研究，以确定与时间分辨荧光的兼容性。我们的结果表明，在几种聚甲基丙烯酸甲酯（PMMA）基板中，使用适当的压印条件且对观察到的衰减具有低背景荧光贡献时，Plexiglas品牌在压印特征方面的复制误差最小。对近红外染料标记的寡核苷酸进行分离，以确定在微芯片分离过程中Cy5.5（τf = 930 ps）和IRD700（τf = 851 ps）标记染料之间荧光寿命区分的适用性。这些染料用于标记M13mp18单链DNA模板的T片段（胸腺嘧啶）。DNA梯在9.5厘米长的蛇形通道中、4%线性聚丙烯酰胺凝胶（LPA）凝胶基质中、130 V/cm的电场下、50摄氏度的温度下进行电泳。电泳图谱显示，尽管由于聚合物微器件中潜在的溶质-壁相互作用或器件的电渗流（EOF）特性，在电泳图谱中难以实现单碱基对分辨率，但寿命可以在超过450个碱基的情况下准确读取。尽管进样量较低，但电泳图谱中各个条带的相对标准偏差与使用毛细管凝胶电泳获得的结果相似。
