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.
Single cell analytics allows quantitative investigation of single biological cells from a structural, functional and proteomics point of view and opens possibilities to a novel unamplified cell analysis inherently insensitive to ensemble-averaging, cell-cycle or cell-population effects. We report on three different experimental methods and their application to cellular systems with single molecule sensitivity at the single cell level. Firstly, atomic force microscopy (AFM) can be used to elucidate the surface structure of living bacteria down to the nanometer scale where identification of irregular surface areas and 2D-arrays of regular protein s-layers is possible. Secondly, single cell manipulation and probing experiments with optical tweezers (OT) force spectroscopy allows quantitative identification of individual recognition events of membrane bound receptors. And thirdly, a novel, single cell analysis for protein fingerprinting in structured microfluidic device format will allow a future (label-free) on-chip electrophoretical protein separation of single cells without preamplification.
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