Continuous-flow pI-Based Sorting of Proteins and Peptides in a Microfluidic Chip using Diffusion Potential
Yong-Ak Song
Sponsorship: NSF CAREER Award (CTS-0347348), KIST-IMC, CSBi/Merck Fellowship
In this work, we have developed a simple microfluidic chip that can sort biomolecules based on their isoelectric point (pI) values in a simple buffer system. The new method differs from previous approaches such as transverse isoelectric focusing [1] or free-flow electrophoresis [2] in that this process involves no external power supply and no special ampholyte. Instead, we utilize the diffusion potential generated by the diffusion of different buffer ionic species in-situ at the laminar flow junction. The use of diffusion potential in microfluidics was previously demonstrated with the mass transport of dye molecules between the two streams in [3]. However, they did not explicitly demonstrate a separation of two species. In our device , we establish a laminar flow junction between two buffers with different pH and concentrations. A potential gradient is developed across the liquid junction, generating high enough electric field to mobilize and to collect biomolecules at the boundary when their pI values fall between the two buffer pH values. The computational modeling shows a decreasing potential gradient from 17.1 V/cm to 6.9 V/cm along the 2 mm long micro channel (20 µm deep, 100 µm wide) , as the concentration gradient becomes shallower toward the end of the channel due to mixing (Figure 1). In our initial experiment, two pI-markers (Figure 2) as well as two proteins were successfully sorted in this device, with a flow rate of 5 ~10 µL/min. To characterize the accuracy of this pI-based sorting process, we tested sorting behavior of the device by changing the pH value of the sample buffer in 0.1 pH step. It was shown that a peptide can be sorted into a different output stream with a ~0.1 pH unit resolution. We are currently working on the development of new buffer systems as well as on the hybrid approach with a superimposed external electric field to increase the sorting efficiency and resolution. Once fully developed , it can potentially be a pI-based sample fractionation tool for proteomic analysis of complex biomolecule samples.
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Figure 1. 3D steady-state concentration distribution in a 2 mm long microfluidic channel with a concentration ratio of 200. Based on this concentration distribution, the diffusion potential as well as the potential gradient can be calculated. |
Figure 2. Schematic view of the pI-based sorting process and separation of two pI markers with pI values of 5. 1 and 7 .2 using diffusion potential at a concentration difference of 2 00 and at a flow rate of 10 m l/min. |
References
- Macounova, K., Cabrera, C. R. & Yager, P. "Concentration and separation of proteins in microfluidic channels on the basis of transverse IEF," Analytical Chemistry 73, 1627–1633 (2001).
- Zhang, C. & Manz, A. "High-speed free-flow elecrophoresis on chip," Analytical Chemistry 75, 5759-5766 (2003).
- Munson, M., Cabrera, C. R. & Yager, P. "Passive electrophoresis in microchannels using liquid junction potentials," Electrophoresis 23, 2642-2652 (2002).
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