Label-free microfluidic free-flow isoelectric focusing, pH gradient sensing and near real-time isoelectric point determination of biomolecules and blood plasma fractions.
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.
Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany. nagl@chemie.uni-leipzig.de.
Elisabeth
E
Poehler
Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany. nagl@chemie.uni-leipzig.de.
Christin
C
Herzog
Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany. nagl@chemie.uni-leipzig.de.
Carsten
C
Lotter
Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany. nagl@chemie.uni-leipzig.de.
Simon A
SA
Pfeiffer
Institut für Analytische Chemie und Lebensmittelchemie, Technische Universität Graz, Stremayrgasse 9/III, 8010 Graz, Österreich, Austria.
Daniel
D
Aigner
Institut für Analytische Chemie und Lebensmittelchemie, Technische Universität Graz, Stremayrgasse 9/III, 8010 Graz, Österreich, Austria.
Torsten
T
Mayr
Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany. nagl@chemie.uni-leipzig.de.
Stefan
S
Nagl
D000667:Ampicillin / Q000302:isolation & purification*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification*; D001798:Blood Proteins / Q000302:isolation & purification*; D046209:Electrophoresis, Microchip / Q000295:instrumentation*; D004867:Equipment Design; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation; D007526:Isoelectric Point; D007768:Lactalbumin / Q000302:isolation & purification*; D007782:Lactoglobulins / Q000302:isolation & purification*; D015242:Ofloxacin / Q000302:isolation & purification*
D000900:Anti-Bacterial Agents; D001798:Blood Proteins; D007782:Lactoglobulins; D000667:Ampicillin; D007768:Lactalbumin; D015242:Ofloxacin