Microfluidic High-Resolution Free-Flow Isoelectric Focusing

Abstract

A microfluidic free-flow isoelectric focusing glass chip for separation of proteins is described. Free-flow isoelectric focusing is demonstrated with a set of fluorescent standards covering a wide range of isoelectric points from pH 3 to 10 as well as the protein HSA. With respect to an earlier developed device, an improved microfluidic FFE chip was developed. The improvements included the usage of multiple sheath flows and the introduction of preseparated ampholytes. Preseparated ampholytes are commonly used in large-scale conventional free-flow isoelectric focusing instruments but have not been used in micromachined devices yet. Furthermore, the channel depth was further decreased. These adaptations led to a higher separation resolution and peak capacity, which were not achieved with previously published free-flow isoelectric focusing chips. An almost linear pH gradient ranging from pH 2.5 to 11.5 between 1.2 and 2 mm wide was generated. Seven isoelectric focusing markers were successfully and clearly separated within a residence time of 2.5 s and an electrical field of 20 V mm-1. Experiments with pI markers proved that the device is fully capable of separating analytes with a minimum difference in isoelectric point of Delta(pI) = 0.4. Furthermore, the results indicate that even a better resolution can be achieved. The theoretical minimum difference in isoelectric point is Delta(pI) = 0.23 resulting in a peak capacity of 29 peaks within 1.8 mm. This is an 8-fold increase in peak capacity to previously published results. The focusing of pI markers led to an increase in concentration by factor 20 and higher. Further improvement in terms of resolution seems possible, for which we envisage that the influence of electroosmotic flow has to be further reduced. The performance of the microfluidic free-flow isoelectric focusing device will enable new applications, as this device might be used in clinical analysis where often low sample volumes are available and fast separation times are essential.

Mathematical and numerical model to study two-dimensional free flow isoelectric focusing

“…4 However, even though their work has demonstrated the feasibility of FFIEF in a microchip, it was severely restricted to low applied electric fields because of bubble formation at the electrode surfaces. To circumvent the bubble problem, Kohlheyer et al 5,6 introduced a modified FFIEF micro-device by adopting anodic and cathodic sheath flows. In their experimental work, a linear pH gradient was developed and the separation of pI makers was accomplished.…”

Fast Electrophoretic Separation Optimization Using Gradient Micro Free-Flow Electrophoresis

“…[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.…”

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

“…In contrast to actively cooled conventional FFE systems the shallower separation region of µ-FFE systems favors fast heat dissipation reducing this type of sample distortion and usually also much lower electrical currents are involved. Experiments have confirmed that µ-FFE separations could be performed without noticeable influence of Joule heating applying electrical field strengths of up to 60 V mm -1 [35,36] with an exception given by [37].…”

“…At elevated fluid velocities and especially during FFIEF the membranes eventually broke. An improved version of this FFE chip was developed and used to achieve an increased separation resolution in FFIEF [36]. …”

“…Kohlheyer et al [36] reported later an improved FFIEF chip based on their previous publication. Here, a pre-separated ampholyte buffer was used which reduced the electrical current and the focusing time.…”