DNA analysis on microfabricated electrophoretic devices with bubble cells

Abstract

Microfluidic devices with bubble cells have been fabricated on poly(methyl methacrylate) (PMMA) plates and have been employed for the analysis of DNA using polyethylene oxide (PEO) solutions. First, the separation channel was fabricated using a wire-imprinting method. Then, wires with greater sizes or a razor blade glued in a polycarbonate plate was used to fabricate bubble cells, with sizes of 190-650 microm. The improvements in resolution and sensitivity have been achieved for large DNA (> 603 base pair, bp) using such devices, which depend on the geometry of the bubble cell. The main contributor for optimal resolution is mainly due to DNA migration at lower electric field strengths inside the bubble cell. On the other hand, slight losses of resolution for small DNA fragments have been found mainly due to diffusion, supported by the loss of resolution when separating two small solutes. With a bubble cell of 75 microm (width) x 500 microm (depth), the sensitivity improvement up to 17-fold has been achieved for the 271 bp fragment in the separation of PhiX-174/HaeIII DNA restriction fragments. We have also found that a microfluidic device with a bubble cell of 360 microm x 360 microm is appropriate for DNA analysis. Such a device has been used for separating DNA ranging from 8 to 2176 bp and polymerase chain reaction (PCR) products amplified after 30 cycles, with rapidity and improvements in the sensitivity as well as resolution.

Ring-down absorption spectroscopy for analytical microdevices

“…A conceptually simple way to increase the absorption path is the introduction of ÔZ-cellsÕ [12,13], ÔU-cellsÕ [14] or bubble cells [15,16] into the microchannel (Fig. 1) While these cells are straightforward to implement in a microfluidic device -although maybe less straightforward to interface to a spectrometer -the enhanced absorption path comes at the cost of reduced temporal resolution and therefore loss in separating power.…”

Low Viscous Separation Media for Genomics and Proteomics Analysis on Microchip Electrophoresis System

“…Several DNA approaches has been performed on chip such as mutation analysis, SNPs analysis, forensic human identification, and genotyping, [55][56][57][58][59][60][61][62] therefore a sensitive, efficient and low viscous separation media has become increasingly important for DNA separation on chip. Below several low viscous separation media has been introduced for analysis on chip.…”

“…[11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] Several types of chips has been introduced for the postgenomic researches such as DNA/RNA chips [28][29][30][31] and the serial analysis of gene expression. [32][33][34][35] The viscosity of the separation media is one important factor for chip separation since the bounded microfluidic devices can not withstand high loading pressure.…”

Modification of poly(methyl methacrylate) microchannels for highly efficient and reproducible electrophoretic separations of double-stranded DNA

“…Polymer solutions are advantageous over cross-linked gels, including relatively low viscosity, ease of preparation, and flexibility. Common polymer solutions used for DNA sequencing, forensic applications, and the analysis of polymerase chain reaction (PCR) products in MCE are prepared from linear polymers, including cellulose and its derivatives, linear poly(acrylamide) (LPA), poly(ethylene oxide) (PEO), or poly(vinyl pyrrolidone) (PVP) [8][9][10][11][12].…”

“…The aim of this study is to develop an effective way to coat the PMMA microchannel surface in order to conduct DNA separation using low-viscosity PEO(GNP) solutions. Based on our previous results [12,24], we fabricated multilayer-coated PMMA chips. In this paper, we demonstrated reproducible, high-resolving, and high-speed DNA separations using a 2-(PEO-PVP)-PEO(GNP) PMMA chip with a 75 m channel filled with 0.75% PEO containing 56nm GNPs (viscosity = 158 cP).…”