Malgorzata A Witek | PubMed Health

PubMed2006

Purification and preconcentration of genomic DNA from whole cell lysates using photoactivated polycarbonate (PPC) microfluidic chips.

We discuss the use of a photoactivated polycarbonate (PPC) microfluidic chip for the solid-phase, reversible immobilization (SPRI) and purification of genomic DNA (gDNA) from whole cell lysates. The surface of polycarbonate was activated by UV radiation resulting in a photo-oxidation reaction, which produced a channel surface containing carboxylate groups. The gDNA was selectively captured on this photoactivated surface in an immobilization buffer, which consisted of 3% polyethylene glycol, 0.4 M NaCl and 70% ethanol. The methodology reported herein is similar to conventional SPRI in that surface-confined carboxylate groups are used for the selective immobilization of DNA; however, no magnetic beads or a magnetic field are required. As observed by UV spectroscopy, a load of approximately 7.6 +/- 1.6 microg/ml of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be 85 +/- 5%. The immobilization and purification assay using this PPC microchip could be performed within approximately 25 min as follows: (i) DNA immobilization approximately 6 min, (ii) chip washout with ethanol 10 min, and (iii) drying and gDNA desorption approximately 6 min. The PPC microchip could also be used for subsequent assays with no substantial loss in recovery, no observable carryover and no need for 'reactivation' of the PC surface with UV light.

Buffers↗

PubMed2004

Cell transport via electromigration in polymer-based microfluidic devices.

Electrokinetic transport of Escherichia coli and Saccharomyces cerevisiae (baker's yeast) cells was evaluated in microfluidic devices fabricated in pristine and UV-modified poly(methyl methacrylate)(PMMA) and polycarbonate (PC). Chip-to-chip reproducibility of the cell's apparent mobilities (micro(app)) varied slightly with a RSD of approximately 10%. The highest micro(app) for baker's yeast cells was observed in UV-modified PC with 0.5 mM PBS (pH = 7.4), and the lowest was measured in pristine PMMA with 20 mM PBS (pH = 7.4). Baker's yeast in all devices migrated toward the cathode because of their smaller electrophoretic mobility compared to the EOF. In 0.5 mM and 1 mM PBS, E. coli cells migrated toward the anode in all cases, opposite to the direction of the EOF due to their larger electrophoretic mobility. E. coli cells in 20 mM PBS migrated toward the cathode, which indicated that the electrophoretic mobility of E. coli cells decreased at higher ionic strengths. Observed differential migrations of E. coli and baker's yeast cells in appropriately prepared polymer microchips were used as the basis for selective introduction into microfluidic devices of only one type of cell. As a working model, experiments were performed with E. coli and RBCs (red blood cells). RBCs migrated toward the cathode in pristine PMMA with 1 mM and 20 mM PBS (pH = 7.4), opposite to the direction of the E. coli cells. By judicious choice of the buffer concentration in which the cell suspension was prepared and the polymer material, RBCs or E. coli cells were selectively introduced into the microdevice, which was monitored via laser backscatter signals.

Cells↗