Towards single molecule analysis in PDMS microdevices: from the detection of ultra low dye concentrations to single DNA molecule studies

Abstract

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.

Recent developments in capillary electrophoresis and capillary electrochromatography of peptides

“…With special designs of detection cells, such as liquid sheath-flow cuvettes and integrated separation and detection devices, LIF detection has a potential to achieve the detection limit of few or even of a single molecule [209][210][211]. The disadvantage of the LIF detection of peptides is the necessity of their derivatization by fluorogenic label.…”

CE – a multifunctional application for clinical diagnosis

“…amperometric or conductivity detection represent interesting expansions of the technical applicability of CE, to our knowledge their clinical application is rather rare. In contrast, CE-LIF techniques are widely applied in clinical investigations, especially in DNA/RNA screenings [47,48,65,66]. The LOD of indirect absorbance detector depends on the absorbance of the buffer and the stability of the intensity of transmitted light.…”

Mesoscopic simulations of electroosmotic flow and electrophoresis in nanochannels

“…In free solution, the electrophoretic mobility of high molecular weight polyelectrolytes is length independent [3]. Electrophoretic separation methods therefore often introduce the samples into micro-or nanostructured environments, e.g., disordered gels (in gel electrophoresis), or structured microchannels [3,4,5,6,7,8,9,10,11,12,13]. The migration behavior of the molecules in such a setup results from a complex interplay of electrostatics, hydrodynamics, and confinement effects on the molecules, and modeling all of this in full detail is computationally challenging.…”