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

Elisabeth Poehler,Carsten Lotter,Torsten Mayr,Daniel Aigner,Christin Herzog,Simon A Pfeiffer,Stefan Nagl

doi:10.1039/c5an01345c

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.

Highlights

Owing to the complexity of biological matrices, separation techniques are very important for the pretreatment of proteomic samples.3 2D gel electrophoresis is often used for proteomic analysis and enables preparative protein purification.[4,5]
A microscopic setup was built for deep UV fluorescence detection of the electrophoretic separation and simultaneous observation of the pH gradient in the NIR and is described below
The setup was implemented for the combination of two imaging excitation light sources, one for the sensor layer and 7498 | Analyst, 2015, 140, 7496–7502

Summary

Introduction

Optical fluorescent and luminescent chemical sensors allow fast, sensitive and selective analysis of different parameters.[21,22] Fluorescent and luminescent pH sensors are widely applied in the monitoring of bioprocesses.[23]. We apply label-free detection via deep UV fluorescence excitation in μFFIEF and combine it with the online observation of the pH gradient in the isoelectric focusing of proteins, antibiotics and blood plasma To achieve this we fabricated a microfluidic quartz FFIEF chip with an integrated near infrared (NIR) fluorescent pH sensor layer. We used an inverted fluorescence microscope (IX-71, Olympus, Tokyo, Japan) which was equipped with a 660 nm LED (M660L3, Thorlabs, Dachau, Germany) as the light source for the integrated pH sensor, controlled via a high power LED driver (DC2100, Thorlabs, Dachau, Germany), a Fluar 2.5×/0.12 objective (Zeiss, Jena, Germany) and a PRO EM 512 EMCCD camera system (Princeton Instruments, Warstein, Germany) for the optical observation of the pH gradient and the separated analytes. For 266 nm excitation of the intrinsic fluorescence of the biomolecules we employed a 140 mW, 20 MHz picosecond Nd:YVO4 laser (Cougar, Time-Bandwidth Products, Zurich, Switzerland)

Results and discussion

Literature pI

Conclusions