Improved native UV laser induced fluorescence detection for single cell analysis in poly(dimethylsiloxane) microfluidic devices.
Single cell analytics is a key method in the framework of proteom research allowing analyses, which are not subjected to ensemble-averaging, cell-cycle or heterogeneous cell-population effects. Our previous studies on single cell analysis in poly(dimethylsiloxane) microfluidic devices with native label-free laser induced fluorescence detection [W. Hellmich, C. Pelargus, K. Leffhalm, A. Ros, D. Anselmetti, Electrophoresis 26 (2005) 3689] were extended in order to improve separation efficiency and detection sensitivity. Here, we particularly focus on the influence of poly(oxyethylene) based coatings on the separation performance. In addition, the influence on background fluorescence is studied by the variation of the incident laser power as well as the adaptation of the confocal volume to the microfluidic channel dimensions. Last but not least, the use of carbon black particles further enhanced the detection limit to 25 nM, thereby reaching the relevant concentration ranges necessary for the label-free detection of low abundant proteins in single cells. On the basis of these results, we demonstrate the first electropherogram from an individual Spodoptera frugiperda (Sf9) cell with native label-free UV-LIF detection in a microfluidic chip.
Hellmich
Wibke
W
Experimental Biophysics and Applied Nanoscience, Physics Department, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany.
Greif
Dominik
D
Pelargus
Christoph
C
Anselmetti
Dario
D
Ros
Alexandra
A
D000818:Animals; D002460:Cell Line; D004129:Dimethylpolysiloxanes / Q000737:chemistry*; D046209:Electrophoresis, Microchip / Q000295:instrumentation / Q000379:methods*; D005260:Female; D005453:Fluorescence; D007834:Lasers; D015203:Reproducibility of Results; D018411:Spodoptera; D014466:Ultraviolet Rays
D004129:Dimethylpolysiloxanes