ABSTRACT
In the present study, we introduce two-photon excitation at 532 nm for label-free fluorescence detection in chip electrochromatography. Two-photon excitation at 532 nm offers a promising alternative to one-photon excitation at 266 nm, as it enables the use of economic chip materials instead of fused silica. In order to demonstrate these benefits, one-photon and two-photon induced fluorescence detection are compared in different chip layouts and materials with respect to the achievable sensitivity in the detection of polycyclic aromatic hydrocarbons (PAHs). Customized chromatography chips with cover or bottom slides of different material and thickness are produced by means of a rapid prototyping method based on liquid-phase lithography. The design of thin bottom chips (180 µm) enables the use of high-performance immersion objectives with low working distances, which allows one to exploit the full potential of two-photon excitation for a sensitive detection. The developed method is applied for label-free analysis of PAHs separated on a polymer monolith inside polymer glass sandwich chips made from fused silica or soda-lime glass. The obtained limits of detection range from 40 nM to 1.95 µM, with similar sensitivities in fused silica thin bottom chips for one-photon and two-photon excitation. In deep-UV non- or less-transparent devices two-photon excitation is mandatory for label-free detection of aromatics with high sensitivity.
ABSTRACT
A current challenge in continuous flow chemistry using microfluidic devices is the combination with a micropreparative separation technique for online product clean-up and analysis. Herein we describe an approach for seamless on-chip integration of continuous flow reactors with a downstream separation functionality based on free-flow electrophoresis. A prototype device was fabricated via liquid phase lithography in a one-step procedure. This proof of concept was successfully validated by performing a model reaction of amino acids with ortho-phthaldialdehyde and continuous separation of the resulting products via micro free-flow zone electrophoresis.
ABSTRACT
We immobilized adherent human embryonic kidney (HEK) cells--which are able to trace adenosine triphosphate (ATP)--inside a microfluidic free-flow electrophoresis (µFFE) chip in order to develop an integrated device combining separation and biosensing capabilities. HEK 293 cells loaded with fluorescent calcium indicators were used as a model system to enable the spatially and temporally resolved detection of ATP. The local position of a 20 µM ATP stream was successfully visualized by these cells during free-flow electrophoresis, demonstrating the on-line detection capability of this technique towards native, unlabeled compounds.
Subject(s)
Adenosine Triphosphate/analysis , Electrophoresis/instrumentation , Microfluidic Analytical Techniques/instrumentation , Biosensing Techniques/instrumentation , Cell Adhesion , Electrophoresis/methods , Equipment Design , Fluorescent Dyes , HEK293 Cells , Humans , Microfluidic Analytical Techniques/methodsABSTRACT
Functional microfluidic free-flow electrophoresis chips with integrated fluorescent pH sensors are presented. Polyethylene glycol based structures were fabricated that allowed for integration of both functions on a single microchip. Microchips were applied in free-flow isoelectric focusing of model compounds and proteins with on-line monitoring of pH during microscale electrophoretic separation.
Subject(s)
Electrophoresis, Microchip/instrumentation , Isoelectric Focusing/instrumentation , Animals , Cattle , Conalbumin/isolation & purification , Equipment Design , Fluorescence , Fluorescent Dyes/analysis , Hydrogen-Ion Concentration , Polyethylene Glycols/chemistry , Serum Albumin, Bovine/isolation & purificationABSTRACT
We present a fast and versatile method to produce functional micro free-flow electrophoresis chips. Microfluidic structures were generated between two glass slides applying multistep liquid-phase lithography, omitting troublesome bonding steps or cost-intensive master structures. Utilizing a novel spacer-less approach with the photodefinable polymer polyethyleneglycol dimethacrylate (PEG-DA), microfluidic devices with hydrophilic channels of only 25 µm in height were generated. The microfluidic chips feature ion-permeable segregation walls between the electrode channels and the separation bed and hydrophilic surfaces. The performance of the chip is demonstrated by free-flow electrophoretic separation of fluorescent xanthene dyes and fluorescently labeled amino acids.
