Free-flow zone electrophoresis and isoelectric focusing using a microfabricated glass device with ion permeable membranes

Abstract

This paper describes a microfabricated free-flow electrophoresis device with integrated ion permeable membranes. In order to obtain continuous lanes of separated components an electrical field is applied perpendicular to the sample flow direction. This sample stream is sandwiched between two sheath flow streams, by hydrodynamic focusing. The separation chamber has two open side beds with inserted electrodes to allow ventilation of gas generated during electrolysis. To hydrodynamically isolate the separation compartment from the side electrodes, a photo-polymerizable monomer solution is exposed to UV light through a slit mask for in situ membrane formation. These so-called salt-bridges resist the pressure driven fluid, but allow ion transport to enable electrical connection. In earlier devices the same was achieved by using open side channel arrays. However, only a small fraction of the applied voltage was effectively utilized across the separation chamber during free-flow electrophoresis and free-flow isoelectric focusing. Furthermore, the spreading of the carrier ampholytes into the side channels resulted in a very restricted pH gradient inside the separation chamber. The chip presented here allows at least 10 times more efficient use of the applied potential and a nearly linear pH gradient from pH 3 to 10 during free-flow isoelectric focusing could be established. Furthermore, the application of hydrodynamic focusing in combination with free-flow electrophoresis can be used for guiding the separated components to specific chip outlets. As a demonstration, several standard fluorescent markers were separated and focused by free-flow zone electrophoresis and by free-flow isoelectric focusing employing a transversal voltage of up to 150 V across the separation chamber.

Fast Electrophoretic Separation Optimization Using Gradient Micro Free-Flow Electrophoresis

“…However, because of the different linear velocities within the channels, the gradient reaches the outlet of the electrode channel ∼16 times faster than in the separation channel. As a result, a concentration mismatch (CM) is created between the channels and is a function of the flow rate and the gradient speed: (6) where t g is the gradient time, L is the length of the channels, and v EC and v SC are the linear velocities in the electrode and separation channels, respectively. Thus at a standard flow rate of 0.5 mL/ min where linear velocities in the electrode and separation channels are 2.6 cm/s and 0.16 cm/s, respectively, the maximum temporal difference over the 2.5 cm length of the channel is ∼15 s. In terms of the 5 min gradient run during the experiments described herein, this corresponds to a modest 5% difference in buffer composition between the electrode and separation channel at the outlet of the device.…”

“…[4][5][6][18][19][20] In early designs two major problems were encountered. Inefficient removal of electrolysis products, manifested as bubbles, from the electrode channels degraded separations.…”

“…4 Also, channel designs used to minimize the effects of the bubbles reduced the electric field applied in the separation channel. 5,6 These problems have since been surmounted using methods such as a two-depth channel design 1 and capacitive * To whom correspondence should be addressed. E-mail: bowser@umn.edu.…”

Microfluidic free-flow electrophoresis for proteomics-on-a-chip

“…Kohlheyer et al [34] presented a µ-FFE glass chip with photo-polymerized acrylamide membranes. This device was fabricated by using two wafers of Borofloat glass, one containing the 15 µm high separation chamber as well as inlet and outlets, as shown in Fig.…”

“…The variance due to the sample injection width w i is usually expressed by [33] Reducing the sample injection width is often easier in microfluidic systems than in larger fluidic systems for example by the precise control of the neighboring laminar flow streams causing hydrodynamic focusing of the sample as for example shown in reference [34].…”