ABSTRACT
Chip-integrated, two-dimensional high performance liquid chromatography is introduced to monitor enantioselective continuous micro-flow synthesis. The herein described development of the first two-dimensional HPLC-chip was realized by the integration of two different columns packed with reversed-phase and chiral stationary phase material on a microfluidic glass chip, coupled to mass spectrometry. Directed steering of the micro-flows at the joining transfer cross enabled a heart-cut operation mode to transfer the chiral compound of interest from the first to the second chromatographic dimension. This allows for an interference-free determination of the enantiomeric excess by seamless hyphenation to electrospray mass spectrometry. The application for rapid reaction optimization at micro-flow conditions is exemplarily shown for the asymmetric organocatalytic continuous micro-flow synthesis of warfarin.
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.
Subject(s)
Fluorescent Dyes/chemistry , Isoelectric Focusing/methods , Lab-On-A-Chip Devices , Microscopy, Fluorescence/methods , Models, Molecular , Peptide Fragments/chemistry , Proteins/chemistry , Animals , Humans , Hydrogels , Isoelectric Focusing/instrumentation , Isoelectric Point , Microscopy, Fluorescence/instrumentation , Peptide Fragments/analysis , Peptide Fragments/isolation & purification , Physalaemin/chemistry , Physalaemin/metabolism , Protein Hydrolysates/chemistry , Proteins/analysis , Proteins/isolation & purification , Stereolithography , Trypsin/metabolismABSTRACT
A pH probe with a microsecond luminescence lifetime was obtained via covalent coupling of 6-carboxynaphthofluorescein (CNF) moieties to ruthenium-tris-(1,10-phenanthroline)(2+). The probe was covalently attached to amino-modified poly-(2-hydroxyethyl)methacrylate (pHEMA) and showed a pH-dependent FRET with luminescence lifetimes of 681 to 1260 ns and a working range from ca. pH 6.5 to 9.0 with a pKa of 7.79 ± 0.14. The pH sensor matrix was integrated via spin coating as ca. 1- to 2-µm-thick layer into "CytoCapture" cell culture dishes of 6 mm in diameter. These contained a microcavity array of square-shaped regions of 40 µm length and width and 15 µm depth that was homogeneously coated with the pH sensor matrix. The sensor layer showed fast response times in both directions. A microscopic setup was developed that enabled imaging of the pH inside the microchamber arrays over many hours. As a proof of principle, we monitored the pH of Escherichia coli cell cultures grown in the microchamber arrays. The integrated sensor matrix allowed pH monitoring spatially resolved in every microchamber, and the differences in cell growth between individual chambers could be resolved and quantified.
Subject(s)
Hydrogen-Ion Concentration , Escherichia coli/growth & development , Escherichia coli/metabolism , Fluorescence Resonance Energy Transfer , Luminescence , Molecular Probes , Spectrometry, Mass, Electrospray IonizationABSTRACT
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 ß-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.
