Continuous on-chip fluorescence labelling, free-flow isoelectric focusing and marker-free isoelectric point determination of proteins and peptides

Abstract

We present a microfluidic platform that contains a micro flow reactor for on-chip biomolecule labelling that is directly followed by a separation bed for continuous free-flow electrophoresis and has an integrated hydrogel-based near-infrared fluorescent pH sensor layer. Using this assembly, labelling of protein and peptide mixtures, their separation via free-flow isoelectric focusing and the determination of the isoelectric point (pI) of the separated products via the integrated sensor layer could be carried out within typically around 5 minutes. Spatially-resolved immobilization of fluidic and sensing structures was carried out via multistep photolithography. The assembly was characterized and optimized with respect to their fluidic and pH sensing properties and applied in the IEF of model proteins, peptides and a tryptic digest from physalaemine. We have therefore realized continuous sample preparation and preparative separation, analyte detection, process observation and analyte assignment capability based on pI on a single platform the size of a microscope slide.

Recent advances in microfluidic sample preparation and separation techniques for molecular biomarker analysis: A critical review

“…Herzog et al [48] developed an integrated microfluidic device on a glass substrate for electrokinetic labeling and separation of peptides and proteins. The integrated device had a reactor for fluorescent labeling with Atto 425, a separation compartment for free flow isoelectric focusing, and a pH sensor layer to calibrate pI values.…”

Isoelectric Point Separations of Peptides and Proteins

“…Complementary approaches have been described particularly in the development of new devices for electrophoretic separations. Herzog et al described the generation of an on-chip reactor to fluorescently label proteins prior to separation according to charge [80]. In an effort to perform isoelectric point based fractionation without carrier ampholytes or isoelectric buffers, Chen and colleagues described a capillary method in which sample pH and the pH of the anolyte and catholyte solutions are tailored to perform fractionation steps according to charge and, therefore, pI [81].…”

Micro free‐flow isoelectric focusing with integrated optical pH sensors

“…This, however, allowed the implementation of an on‐chip precolumn labeling procedure prior to the FFIEF. Thereby this microfluidic chip could be employed for flow fluorescence labeling, FFIEF with integrated pH sensing as well as pI determination at the same time .…”

“…(A) μFFIEF of the peptides endothelin (En), oxytocin (Ox), leucine‐enkephalin (LE) and neurotensin (Ne) with integrated pH monitoring, (B) Isoelectric focusing of the proteins lactalbumin (La), lactoglobulin (Lg) and myoglobin (My), (C) separation of the products from a tryptic digest of physalaemin. Adapted from with permission from The Royal Society of Chemistry.…”