Protein separation and surfactant control of electroosmotic flow in poly(dimethylsiloxane)-coated capillaries and microchips.

A thermally pyrolyzed poly(dimethylsiloxane) (PDMS) coating intended to prevent surface adsorption during capillary electrophoretic (CE) [Science 222 (1983) 266] separation of proteins, and to provide a substrate for surfactant adsorption for electroosmotic mobility control was prepared and evaluated. Coating fused-silica capillaries or glass microchip CE devices with a 1% solution of 100 cSt silicone oil in CH2Cl2, followed by forced N2 drying and thermal curing at 400 degrees C for 30 min produced a cross-linked PDMS layer. Addition of 0.01 to 0.02% Brij 35 to a 0.020 M phosphate buffer gave separations of lysozyme, cytochrome c, RNase, and fluorescein-labeled goat anti-human IgG Fab fragment. Respective plates/m typically obtained at 20 kV (740 V cm(-1)) were 2, 1.5, 1.25, and 9.4-10(5). In 50 mM ionic strength phosphate, 0.01% Brij 35 running buffer, the electroosmotic flow observed was about 25% of that in a bare capillary, and showed no pH dependence between pH 6.3-8.2. Addition of sodium dodecylsulfate (SDS) or cetyltrimethylammonium bromide (CTAB) to this running buffer allowed ready control of electroosmotic mobility, mu(eo). Concentrations of SDS between 0.005 to 0.1% resulted in mu(eo) ranging from 3 to 5 x 10(-4) cm2 V(-1) s(-1). Addition of 1 to 2.3 x 10(-4)% (2.7-6.3 microM) CTAB caused flow reversal. CTAB concentrations between 3.5 x 10(-4) and 0.05% (0.0014-1.37 mM) allowed control of mu(eo) between -1 x 10(-4) and -5.0 x 10(-4) cm2 V(-1) s(-1). For both surfactants the added presence of 0.01% Brij 35 provided slowly varying changes in mu(eo) with charged surfactant concentration.

Integrated microfluidic electrophoresis system for analysis of genetic materials using signal amplification methods.

An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), was performed within a microfluidic chip. The presence of DNA from methicillin-resistant Staphylococcus aureus was determined by signal amplification of a specific DNA sequence. The microfluidic device consisted of four channels intersecting to mix the sample and reagents within 55 s, as they were directed toward the reactor coil by electrokinetic pumping. The 160-nL CPT reactor occupied approximately 220 mm2. Gel-free capillary electrophoresis separation of the biotin- and fluorescein-labeled probe from the probe fragments was performed on-chip following the on-chip reaction. An off-chip CPT reaction, with on-chip separation gave a detection limit of 2 fM (0.03 amol) target DNA and an amplification factor of 85,000. Calibration curves, linear at <5% probe fragmentation, obeyed a power law relationship with an argument of 0.5 [target] at higher target DNA concentrations for both on-chip and off-chip CPT reaction and analysis. An amplification factor of 42,000 at 250 fM target (25,000 target molecules) was observed on-chip, but the reaction was approximately 4 times less sensitive than off-chip under the conditions used. Relative SD values for on-chip CPT were 0.8% for the peak migration times, 9% for the area of intact probe peak, and 8% for the fragment/probe peak area ratio.