Demonstration of isoelectric focusing on an etched quartz chip with UV absorption imaging detection†

Abstract

The feasibility of isoelectric focusing (IEF) performed on-chip was demonstrated for the first time via absorption imaging detection. Microchannels on a quartz chip were fabricated using photolithography and a chemical etching process. The separation channels were 40 mm long, 100 µm wide and 10 µm deep, and were coated with linear polyacrylamide to reduce electroosmotic flow. A quartz chip cartridge for IEF was assembled in which two pieces of hollow fiber were glued to the two ends of the separation channel to isolate the electrolytes from the samples. Low molecular mass pI markers and myoglobin were selected as model samples which were mixed with 4% carrier ampholyte solution. Samples were injected into the channel via the connection capillary by pressure. A voltage of 3 kV was applied to perform IEF. The IEF current decreased from about 13.4 to 1.3 µA. The focused zones were monitored in real time by absorption imaging detection at 280 nm. The detection limit was about 0.3 µg ml^–1 or 24 pg for pI marker 6.6, and 30 µg ml^–1 or 2.4 ng for myoglobin with an optical pathlength of 10 µm. Good reproducibility and resolution were obtained for linear polyacrylamide coated channels. The total analysis time was less than 10 min. This imaged chip IEF provides a fast separation technique with quantitative ability and the potential for increasing throughput.