Control of surface properties in microfluidic systems is an indispensable prerequisite for successful bioanalytical applications. Poly(dimethylsiloxane) (PDMS) microfluidic devices are hampered from unwanted adsorption of biomolecules and lack of methods to control electroosmotic flow (EOF). In this paper, we propose different strategies to coat PDMS surfaces with poly(oxyethylene) (POE) molecules of varying chain lengths. The native PDMS surface is pretreated by exposure to UV irradiation or to an oxygen plasma, and the covalent linkage of POE-silanes as well as physical adsorption of a triblock-copolymer (F108) are studied. Contact angle measurements and atomic force microscopy (AFM) imaging revealed homogeneous attachment of POE-silanes and F108 to the PDMS surfaces. In the case of F108, different adsorption mechanisms to hydrophilic and hydrophobic PDMS are discussed. Determination of the electroosmotic mobilities of these coatings in PDMS microchannels prove their use for electrokinetic applications in which EOF reduction is inevitable and protein adsorption has to be suppressed.
Single cell analytics for proteomic analysis is considered a key method in the framework of systems nanobiology which allows a novel proteomics without being subjected to ensemble-averaging, cell-cycle, or cell-population effects. We are currently developing a single cell analytical method for protein fingerprinting combining a structured microfluidic device with latest optical laser technology for single cell manipulation (trapping and steering), free-solution electrophoretical protein separation, and (label-free) protein detection. In this paper we report on first results of this novel analytical device focusing on three main issues. First, single biological cells were trapped, injected, steered, and deposited by means of optical tweezers in a poly(dimethylsiloxane) microfluidic device and consecutively lysed with SDS at a predefined position. Second, separation and detection of fluorescent dyes, amino acids, and proteins were achieved with LIF detection in the visible (VIS) (488 nm) as well as in the deep UV (266 nm) spectral range for label-free, native protein detection. Minute concentrations of 100 fM injected fluorescein could be detected in the VIS and a first protein separation and label-free detection could be achieved in the UV spectral range. Third, first analytical experiments with single Sf9 insect cells (Spodoptera frugiperda) in a tailored microfluidic device exhibiting distinct electropherograms of a green fluorescent protein-construct proved the validity of the concept. Thus, the presented microfluidic concept allows novel and fascinating single cell experiments for systems nanobiology in the future.
Microfluidic and lab-on-a-chip devices have attracted widespread interest in separation sciences and bioanalysis. Recent designs in microfluidic devices extend common separation concepts by exploiting new phenomena for molecular dynamics on a length scale of 10 mum and below, giving rise to novel manipulation tools and nonintuitive phenomena for microseparations. Here, we focus on three very recent developments for bioseparations based on tailored microfluidic systems: Single cell navigation, trapping and steering with subsequent on-chip lysis, protein separation and LIF detection (Section 3.1), then we report dielectrophoretic trapping and separation of large DNA fragments in structured microfluidic devices (Section 3.2). Finally, a paradoxial migration phenomenon based on thermal fluctuations, periodically arranged microchannels and a biased alternating current electric field is presented in Section 3.3.
In this paper, we report on the performance of electrophoretical separation and laser-induced fluorescence (LIF) detection of dyes and fluorescently labeled biomolecules in poly(dimethylsiloxane) (PDMS) microdevices. The dyes fluorescein and fluorescein isothiocyanate (FITC) have been separated effectively in nM concentrations. Fluorescein injections gave linear concentration response in the range from 4 to 100 pM. As ultimate detection sensitivity, 100 fM injected fluorescein was obtained. Further, 100 fM injected fluorescein could be detected. This is to our knowledge the smallest electrokinetically injected dye concentration detected on a microchip. Injection studies of fluorescently labeled avidin revealed a theoretical detection limit of 25 nM for laser-induced fluorescence detection in good agreement with separations in glass chips. Furthermore, the injection of several and even one single DNA molecule using a PDMS cross injector has been demonstrated as well as free solution separation of -and T2-DNA (60 pM each) in periodically structured channels.
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