Microfluidic Free-Flow Isoelectric Focusing with Real-Time pI Determination

  • Stefan NaglEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1906)


Free-flow electrophoresis (FFE) may be used for continuous and preparative separation of a wide variety of biomolecules. Isoelectric focusing (IEF) provides for the separation of compounds according to their isoelectric point (pI). Here we describe a microfluidic chip-based protocol for the fabrication, application, and optical monitoring of free-flow isoelectric focusing (FFIEF) of proteins and peptides on the microscale with optical surveillance of the microscopic pH gradient provided by an integrated pH sensing layer. This protocol may be used with modifications also for the FFIEF of other biomolecules and may serve as template for the fabrication of microfluidic chips with integrated fluorescent or luminescent pH sensor layers for FFE and other applications.

Key words

Free-flow electrophoresis Isoelectric focusing Fluorescent pH sensor Isoelectric point determination Multispectral imaging 


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of ChemistryThe Hong Kong University of Science and TechnologyKowloonChina

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