Microfluidic analysis and parallel confocal detection of single molecules

Abstract: The detection of single molecules using confocal spectroscopy is of major interest and tremendous improvements have been made within the last decade. The underlying basics, however, a pinhole and a single excitation volume element have remained the same as well as the technical limitations. This thesis presents an approach to address and overcome these technological hurdles and to design and implement a confocal system with parallel detection capability that increases the throughput and speeds up the analysis time. Therefore, microfluidic systems, diffractive optical excitation, CMOS (Complementary metal oxide semiconductor)detector arrays and the combination of these novel techniques have been investigated. In order to show the usefulness of these techniques, biomolecular applications like screening of Alzheimer's amyloid-beta peptides and gene expression analysis have been investigated as well. In addition to that, microstructure systems together with confocal spectroscopy were used to degrade DNA on the single molecule level or to enrich biological samples. Knowledge of flow behavior in microstructures is essential when microchannels are used for analytical applications. Fluorescence Correlation Spectroscopy (FCS) was utilized to investigate hydrodynamic pressure driven flow in microchannels with a cross section of 50 x 50 µm2. The flow itself, was detected non-altered or noninvasively, since only tiny (a couple of nanometer in size) dye-molecules were entrained into the fluid. This was sufficient to determine the flow velocities in laminar layers of < 1 µm in size. Further, transport properties were monitored in microchannels with the help of a 1 x 4 diffractive optical element (DOE) induced laser excitation array. A step forward towards a highly integrated parallel confocal fluorescent detection setup has been taken, by introducing CMOS-detector arrays in conjunction with diffractive optical elements (DOEs). A combination of a 2 x 2 DOE and a 2 x 2 CMOS-detector array was utilized in order to see if parallel detection of single molecule is feasible. This detection scheme potentially improves and speeds up the analysis of biological and chemical samples because on-chip processing, data analysis, and a high level of integration could easily be realized. Two individual DOEs were used to build a parallel 2 x 2 cross correlation set-up. This technology has been developed for the determination of the gene expression profile by a novel analytical method based on cross correlation analysis. Here, the long analysis time and the constantly present low concentration of the sample motivated the implementation of such a set-up. Additionally, other analytical methods such as high-throughput screening (HTS), could profit from this method decreasing their analysis time in future. The detection of single molecules in flow has been illustrated and utilized, including investigations on single molecule detection in microchannels using elliptical shaped detection volume elements. With the present systems, Alzheimer's beta-amyloid peptides were analyzed and a feasibility for up-concentration of biological or chemical samples was performed with regards to pl flow switching with external valves. Finally, DNA was degraded on the single molecule level and the cleavage rate of the enzyme was determined.