Simple device for multiplexed electrophoretic separations using gradient elution moving boundary electrophoresis with channel current detection.
A new microfluidic electrophoresis device and technique is described that is designed specifically for multiplexed, high-throughput separations. The device consists of an array of short (3 mm) capillaries connecting individual sample reservoirs to a common buffer reservoir. Each capillary in the array functions as both a separation channel and as a conductivity-based detection cell. The new technique is based upon the recently described gradient elution moving boundary electrophoresis (GEMBE) technique, which uses a combination of an electric field and buffer counterflow to achieve electrophoretic separations in short capillaries or microfluidic channels. A high voltage drives electrophoresis of the sample analytes through each separation channel. At the start of a separation, the bulk counterflow of buffer through the channel is high, and none of the analytes of interest can enter the channel. The counterflow is then gradually reduced until each analyte, in turn, is able to enter the channel where it is detected as a moving boundary or step. With very short capillaries, only one step at a time is present in each capillary, and the electric current through the channels can then be used as the detector signal, without any extra detector hardware. The current vs time signal for each channel is then smoothed and differentiated to produce a set of simultaneous electropherograms. Because there is no light source or other added hardware required for detection, the system is simple and can be easily and inexpensively scaled up to perform large numbers of simultaneous analyses. As a first demonstration, a 16-channel array device is used for high-throughput, time-series measurements of enzyme activity and inhibition.
Ross
David
D
Biochemical Science Division, National Institute of Standards & Technology, Gaithersburg, Maryland 20899, USA.
Kralj
Jason G
JG
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D046209:Electrophoresis, Microchip / Q000295:instrumentation* / Q000379:methods*; D007546:Isoquinolines / Q000494:pharmacology*; D046210:Microfluidic Analytical Techniques; D044085:Microfluidics; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D013449:Sulfonamides / Q000494:pharmacology*
D007546:Isoquinolines; D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D000244:Adenosine Diphosphate; C037642:kemptide; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide