Klaus Dörre

Two-beam cross-correlation: A method to characterize transport phenomena in micrometer-sized structures.

To determine flow properties, namely, the velocity and angle of the flow in microstructured channels, an experimental realization based on fluorescence correlation spectroscopy is described. For this purpose, two micrometer-sized spatially separated volume elements have been created. The cross-correlation signal from these has been recorded and evaluated mathematically. In addition to previous results, two-beam cross-correlation allows for fast and easy determination of even small (down to 200 μm/s) flow velocities, as well as simultaneous measurement of diffusion properties of single dye molecules within a rather short detection time of 5-100 s and an error rate of less than 20%. The spatial flow resolution is around 1-2 μm, limited by the diameter of the volume element. Furthermore, vectorial flow data can be obtained and evaluated. A discussion of the theoretical background and an experimental verification of the theoretical results is performed. The feasibility of fast and easy data processing is shown if the flow time is the only desired information. Possible applications of this precise and simple method are the determination of transportation effects within artificial microstructures for CE and HPLC, fast chemical kinetics, and high-throughput screening.

Towards a general procedure for sequencing single DNA molecules.

In this paper we report on the latest technical advances towards single molecule sequencing, a useful method currently developed especially for fast and easy de novo sequencing. Different approaches for complete labeling of DNA with fluorescent dyes are described. In addition, the experimental set-up for the sequencing process is shown. We demonstrate the ability to purify the buffer and enzyme solutions. Inorganic buffers were purified down to at least 20 fM of remaining fluorescent impurities. The exonuclease buffer solution could be cleaned down to 0.8 pM whereby its full activity was kept. Finally, we show a selection procedure for beads and present the data of a model experiment, in which immobilized DNA is degraded by an exonuclease within a polymethylmethacrylate (PMMA) microstructure. Furthermore, the mathematical processing of the obtained raw data is described. A first complete experimental cycle is shown, combining all preparatory steps which are necessary for single molecule sequencing in microstructures.

Highly efficient single molecule detection in microstructures.

For DNA single molecule sequencing, the complete detection of all dye-labeled monomers which are cleaved off during the sequencing reaction is an essential requirement. In this work we address the feasibility of single molecule detection in microstructures with a confocal multi element set-up. We present statistical data on single molecule recognition and explain a refined data evaluation technique for single molecule burst analysis. From these data the signal-to-noise ratio in microstructures is evaluated as well as the overall detection efficiency. So far, detection efficiencies of single molecule events of up to 60% have been shown in microstructures.

Confocal spectroscopy in microstructures

Confocal instrumentation makes it possible to carry out spectroscopic measurements with a very high signal-to-background ratio. The transit of a single fluorescent molecule through the focal point of the light can be monitored with this method. The particle transport can be observed in transparent microchannels. Examples of fluorescence correlation spectroscopy and single molecule handling in microstructures are discussed.