Nanoliter-scale sample preparation methods directly coupled to PMMA-based microchips and gel-filled capillaries for the analysis of oligonucleotides

摘要

Abstract: We are currently developing miniaturized, chip-based electrophoresis devices fabricated in plastics for the high speed separation of oligonucleotides. One of the principal advantages associated with these devices is their small sample requirements, typically in the nanoliter to sub-nanoliter range. Unfortunately, most standard sample preparation protocols, especially for oligonucleotides, are done off-chip on a microliter-scale. Our work has focused on the development of capillary nano-reactors coupled to micro-separation platforms, such as micro-electrophoresis chips, for the preparation of sequencing ladders and also, PCR reactions. These nano-reactors consist of fused silica capillary tubes (length equals 10 - 20 cm; id equals 20 - 50 micrometer) with fluid pumping accomplished using the electro-osmotic flow generated by the tubes. These reactors were situated in fast thermal cyclers to perform cycle sequencing or PCR amplification of the DNAs. The reactors were interfaced to the micro-electrophoresis chips via capillary connectors micromachined in polymethylmethacrylate (PMMA) using deep X- ray etching (width equals 50 micrometer; depth equals 50 micrometer) and were situated directly on the PMMA-based microchip. This chip also contained an injector, separation channel (length equals 6 cm; width equals 30 micrometers; depth equals 50 micrometers) and a dual fiber optic, near- infrared fluorescence detector. The sequencing nano-reactor used surface immobilized templates attached to the wall via a biotin:streptavidin:biotin linkage produced by PCR using a biotinylated forward primer. Sequencing tracks could be directly injected into gel-filled capillary tubes with minimal degradation in the efficiency of the separation process. The nano-reactor could also be configured to perform PCR reactions by filling the capillary tube with the PCR reagents and template. After thermal cycling, the PCR cocktail could be injected into a capillary tube or a micro-chip device for fractionation. In all cases, the detection of the oligonucleotides was accomplished using ultra-sensitive near- IR fluorescence detection. !33