Microfluidic devices were fabricated on poly(methyl methacrylate) (PMMA) substrate using two small-diameter (79 microm) wires to create a cross impression in plastics softened by low-temperature heating. The resulting channels had a rounded shape and 75 microm in depth. The variability of the channel dimensions was found to be less than 6% from different locations of the same channel and less than 10% between chips. Moreover, the fabricated PMMA chip appeared to sustain an electric field strength up to 300 V/cm without significant Joule heating. The function of resulting devices for electrophoretic injection and separation of a DNA size marker, HaeIII digest of (phiX174, was also characterized. Results indicated that all of the 11 DNA fragments of the size marker could be identified in less than 3 min with relative standard deviations less than 0.4% and 8% for migration time and peak area, respectively. Moreover, with the use of near infrared (IR) dye, fluorescence signals of the higher molecular weight fragments (> 603 bp in length) could be detected at total DNA concentrations as low as 0.1 microg/mL (S/N = 4.2). In conclusion, the performance of wire-imprinted devices on PMMA substrate were comparable to those fabricated by other professional means.

Analysis of DNA fragments by microchip electrophoresis fabricated on poly(methyl methacrylate) substrates using a wire-imprinting method.

Background: Electrophoresis on polymeric rather than glass microstructures is a promising separation method for analytical chemistry. Assays on such devices need to be explored to allow assessment of their utility for the clinical laboratory.

Methods: We compared capillary and plastic microchip electrophoresis for clinical post-PCR analysis of hepatitis C virus (HCV). For capillary electrophoresis (CE), we used a separation medium composed of 10 g/L hydroxypropyl methyl cellulose in Tris-borate-EDTA buffer and 10 μmol/L intercalating dye. For microchip electrophoresis, the HCV assay established on the fused silica tubing was transferred to the untreated polymethylmethacrylate microchip with minimum modifications.

Results: CE resolved the 145-bp amplicon of HCV in 15 min. The confidence interval of the migration time was <3.2%. The same HCV amplicon was resolved by microchip electrophoresis in <1.5 min with the confidence interval of the migration time <1.3%.

Conclusion: The polymer microchip, with advantages that include fast processing time, simple operation, and disposable use, holds great potential for clinical analysis.

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Plastic Microchip Electrophoresis for Analysis of PCR Products of Hepatitis C Virus

Different lengths of antisense DNA are normally separated by Polyacrylamide gelfilled column using capillary electrophoresis. The rigid gel bed, however, has a limited lifetime due to the forming of air bubbles and clogging particularly when a real sample with complex matrix is analyzed. An alternative is to replace the gel with a replenishable polymer solution. In this work, a capillary electrophoresis system using a microcross (10 nL ) connected with four 75 μm i.d. fused-silica capillaries and operated under high-voltage shunting is described to implement the PEG sieving medium for the separation of antisense DNAs. Both the separation and injection capillaries were filled with polyethylene glycol (PEG) in tris-borate buffer and the separation was performed with pinched-mode. The PEG medium was found to resolve the 15–20mers of antisense DNA under a conventional linear configuration. Substantial band broadening, however, was observed when using the cross configuration under the same separation conditions. A better control of the injection time as well as the applied voltage is required to minimize the sample diffusion.

Shin-Hwan Wang

Papers

Electrophoretic Separation of Antisense DNA using Polymer-Solution Filled Capillary by Cross-Injection