Champak Das

Integration of isoelectric focusing with multi-channel gel electrophoresis by using microfluidic pseudo-valves

Two-dimensional (2D) protein separation is achieved in a plastic microfluidic device by integrating isoelectric focusing (IEF) with multi-channel polyacrylamide gel electrophoresis (PAGE). IEF (the first dimension) is carried out in a 15 mm-long channel while PAGE (the second dimension) is in 29 parallel channels of 65 mm length that are orthogonal to the IEF channel. An array of microfluidic pseudo-valves is created for introducing different separation media, without cross-contamination, in both dimensions; it also allows transfer of proteins from the first to the second dimension. Fabrication of pseudo-valves is achieved by photo-initiated, in situ gel polymerization; acrylamide and methylenebisacrylamide monomers are polymerized only in the PAGE channels whereas polymerization does not take place in the IEF channel where a mask is placed to block the UV light. IEF separation medium, carrier ampholytes, can then be introduced into the IEF channel. The presence of gel pseudo-valves does not affect the performance of IEF or PAGE when they are investigated separately. Detection in the device is achieved by using a laser induced fluorescence imaging system. Four fluorescently-labeled proteins with either similar pI values or close molecular weight are well separated, demonstrating the potential of the 2D electrophoresis device. The total separation time is less than 10 minutes for IEF and PAGE, an improvement of 2 orders of magnitude over the conventional 2D slab gel electrophoresis.

Laser-Induced Fluorescence Imaging System for Protein Separations in Microfluidic Devices

This paper describes a laser-induced fluorescence (LIF) detection system for imaging proteins separated in a microfluidic device. The diameter of a laser beam is first increased through a beam expander, and subsequently focused into a line using a cylindrical lens. The resultant laser line is used to image an entire capillary or channel in which protein separation took place. The fluorescence emission is collected with a cooled, scientific grade charge-coupled device (CCD) camera. The detection limit was determined using a series of concentrations of fluorescein solutions. The temporal and spatial effects of photobleaching from laser irradiation were analyzed and the parameters to reduce the effect of photobleaching are discussed. We used the imaging system to demonstrate rapid analysis of proteins using isoelectric focusing.

Rapid Protein Separations in Microfluidic Devices

Abstract : This paper describes fabrication of glass and plastic microfluidic devices for protein separations. Although the long-term goal is to develop a microfluidic device for two-dimensional gel electrophoresis, this paper focuses on the first dimension-isoelectric focusing (IEF). A laser-induced fluorescence (LIF) imaging system has been built for imaging an entire channel in an IEF device. The whole-channel imaging eliminates the need to migrate focused protein bands, which is required if a single-point detector is used. Using the devices and the imaging system, we are able to perform IEF separations of proteins within minutes rather than hours in traditional bench-top instruments.

Fabricating Plastic Microfluidic Devices With Photodefinable Microvalves for Protein Separations

This paper describes the results of fabricating plastic microfluidic devices and creating a microvalve array for protein separation. Plastic devices are selected due to low cost of raw materials, bio-compatibility, and disposability. Although the methods for fabricating plastic devices have appeared in literature, reports typically indicate one set of conditions that yield functional devices. We report a systematic study of fabrication process parameters including compression rate, molding temperature, and the compression force used by a hydraulic press. Their effects on the device thickness, channel dimension, and pattern transfer fidelity will be discussed. In addition, we investigated creating an array of pseudo-microvalves using photodefinable, in situ gel polymerization. The valves were developed for introducing two types of separation media for performing two-dimensional protein separation in a microfluidic device. We also demonstrated rapid protein separation using the mechanism for the first dimension, isoelectric focusing.Copyright