Label-free microfluidic free-flow isoelectric focusing, pH gradient sensing and near real-time isoelectric point determination of biomolecules and blood plasma fractions

Abstract

We demonstrate the fabrication, characterization and application of microfluidic chips capable of continuous electrophoretic separation via free flow isoelectric focussing (FFIEF). By integration of a near-infrared (NIR) fluorescent pH sensor layer under the whole separation bed, on-line observation of the pH gradient and determination of biomolecular isoelectric points (pI) was achieved within a few seconds. Using an optical setup for imaging of the intrinsic fluorescence of biomolecules at 266 nm excitation, labelling steps could be avoided and the native biomolecules could be separated, collected and analysed for their pI. The fabricated microchip was successfully used for the monitoring of the separation and simultaneous observation of the pH gradient during the isoelectric focussing of the proteins α-lactalbumin and β-lactoglobulin, blood plasma proteins and the antibiotics ampicillin and ofloxacin. The obtained pIs are in good agreement with literature data, demonstrating the applicability of the system. Mass spectra from the separated antibiotics taken after 15 minutes of continuous separation from different fractions at the end of the microchip validated the separation via microfluidic isoelectric focussing and indicate the possibility of further on- or off-chip processing steps.

Micro free‐flow isoelectric focusing with integrated optical pH sensors

“…In this study we also collected fractions at the outlets for the antibiotics separation and analyzed them via ESI‐MS. The MS data confirmed the successful separation of the compounds with only a very small residue left of the respective other compound in each fraction and the ability of the platforms to transport the separated compounds to different locations .…”

“…C). The bottom side of the microfluidic chip as well as the (bottom side) microscope optical systems block all deep UV excitation light thereby acting as additional filters and the epi‐illumination stage of the microscope is available for excitation of the pH sensor layer using a 660 nm light emitting diode (LED) and appropriate optics and filters .…”

“…(B) Image of the microfluidic chip on an inverted fluorescence microscope with laser excitation from above and, (C) macroscopic images of the excitation laser light path. Adapted from with permission from The Royal Society of Chemistry.…”

Analysis of antibiotics by CE and CEC and their use as chiral selectors: An update

“…In the last years, microfluidic devices have attracted the attention of researchers in the field of analytical separations due to their inherent characteristics such as very low sample and solvent consumption, short analysis time, and moderately high resolution . Electrophoresis in microchip (MCE) offers some advantages such as high heat dissipation which allows the application of higher electric fields yielding high resolution and short analysis times.…”

Carrier ampholyte‐free free‐flow isoelectric focusing for separation of protein

“…This method takes short time (∼5 min) and does not require pre‐focusing. The other is called natural pH gradient , which is accomplished by pumping a mixed ampholyte solution into the separation chamber to directly establish the pH gradient in the separation chamber. This method takes a long time (>30 min), but has a better focusing effect than the first method.…”