Jens Stephan

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.

Flash-photolysis of 2-(2'-hydroxyphenyl)-3-H-indole. Ground-state keto-enol tautomerization by mutual hydrogen exchange and by proton catalysis

Abstract Efficient excited-state intramolecular proton transfer (ESIPT) in 2-(2′-hydroxy-5′-methylphenyl)-3,3-dimethyl-3H-indole (HBC) leads to the formation of an unstable trans-keto tautomer 1 K tr whose transient absorption can be observed in flash experiments. The assignment of this transient absorption to the trans-keto tautomer is corroborated by the observation that a similar transient is not formed from BHBC, which has been synthesized as a bridged, rotationally blocked counterpart of HBC. In hydrocarbon solvents, two 1 K tr molecules revert by a mutual hydrogen-exchange reaction to the stable enol form of HBC. This second-order reaction is diffusion-controlled and its rate constant is proportional to T /η over a wide range of the solvent viscosity η. In the presence of alcohols (ROH) a first-order, pseudo-monomolecular proton-catalysed re-enolization reaction competes with the second-order reaction. With methanol and ethanol as proton donors it is shown that two donor molecules are involved in this process. The rate of the proton-catalysed reaction increases with decreasing temperatures. A pre-equilibrium between a nonreactive 1 K tr and a reactive 1 K tr ::: (ROH) 2 complex is the reason for this unusual temperature dependence of the first-order 1 K tr decay rate.

Photoisomerizations of the photochromic anil salicylidene-1-naphthylamine in 3-methylpentane

Abstract The results of a nanosecond flash-photolysis study with solutions of salicylidene-1-naphthyl-amine (SN) in 3-methylpentane (3MP) at ambient and at low temperatures are reported. Besides a newly discovered triplet state, two photochromic transients (PCTs) are formed after excited state intramolecular proton-transfer (ESIPT) has taken place. Above 140 K the formation of a quinoid trans-keto tautomer (1Ktr) prevails. At 100 K a second PCT is observed in steady-state experiments which we tentatively assign to a twisted conformer of the keto form of SN. The transient 1Ktr re-enolizes in dry 3MP by second-order double proton-transfer; in the presence of 10−4 M methanol the re-enolization of 1Ktr is proton catalyzed and the lifetime of 1Ktr is shortened by more than two orders of magnitude. The properties of the flexible SN are compared with two other rotationally hindered ‘ESIPT molecules’.

Dual-Color Confocal Fluorescence Spectroscopy and its Application in Biotechnology

In recent years, fluorescence correlation spectroscopy (FCS) has become an attractive analytical tool for the investigation of biomolecular processes at the single molecular level. This method was invented in the early 1970s by groups at Cornell University, Ithaca, N.Y. [9.1,9.2], and at the Karolinska Institute, Stockholm [9.3,9.4]. During the 1990s, modern confocal optics, new dyes as efficient fluorescent probes, sensitive photon detectors, and fast data processing tools have been introduced, mainly by Rigler and Eigen [9.5,9.6]. Due to these improvements, FCS permits the observation of the dynamics of single molecules in real time while they pass an open volume element of less than a femtoliter, i.e. the size of a common bacterial cell. Nowadays, FCS has found its way into several laboratories and companies all over the world as a tool for basic research as well as for industrial applications such as drug screening.