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

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 lambda- and T2-DNA (60 pM each) in periodically structured channels.