Andreas Schulz

New fluorescent dyes in the red region for biodiagnostics.

The increased sensitivity together with the advent of low-cost optical sources and detectors in the visible-near IR region has led us to current efforts to develop new efficient fluorescent labels for biodiagnostics with absorption and emission beyond 600 nm. In view of the general fluorescence decrease with increasing emission wavelength, we investigated the possibility to shift the absorption of rhodamine dyes toward the region 620–670 nm. The hydrophobic nature of all known long-wavelength dyes results in the tendency to form intra- and intermolecular aggregates in hydrophilic solvents, especially in aqueous environment. Due to the aggregation with biological materials, fluorescence quenching of the dyes is often observed. New strategies for prevention of these processes are considered.

New fluorescent labels for time-resolved detection of biomolecules.

New dyes with characteristic fluorescence lifetimes have been developed for bioanalytical applications. Based upon the concept of “multiplex dyes,” we have designed rhodamine dyes with nearly identical absorption and emission spectral characteristics but different fluorescence lifetimes. Extending this principle to applications with laser diodes, new rhodamines with functional groups for covalent coupling of analytes have been developed. The new labels exhibit absortion and fluorescence beyond 600 nm and have a high quantum efficiency, even in aqueous buffer systems.

Molecular mechanics force field parameterization of the fluorescent probe rhodamine 6G using automated frequency matching

Novel single‐molecule fluorescence experimental techniques have prompted a growing need to develop refined computational models of dye‐tagged biomolecules. As a necessary first step towards useful molecular simulations of fluorescence‐labeled biomolecules, we have derived a force field for the commonly used dye, rhodamine 6G (R6G). A novel automated method is used that includes fitting the molecular mechanics potential to both vibrational frequencies and eigenvector projections derived from quantum chemical calculations. The method is benchmarked on a series of aromatic molecules then applied to derive new parameters for R6G. The force field derived reproduces well the crystal structure of R6G.

EFFICIENT DNA SEQUENCING WITH A PULSED SEMICONDUCTOR LASER AND A NEW FLUORESCENT DYE SET

Abstract A new method is presented for automated one-lane four-dye DNA sequencing in capillary gel electrophoresis based on semiconductor technology and a special set of multiplex fluorescent dyes which exhibit similar absorption and emission spectra but different fluorescent lifetimes. The primer sequencing reaction was applied in a confocal optical system. Detection and identification of the differently 5′-labeled primers was done by time-correlated single-photon counting and a specially developed pattern-recognition technique based on the characteristic fluorescence lifetimes of the fluorescent dyes used as labels. Efficient excitation was performed at 630 nm by a short-pulsed semiconductor laser with a repetition rate of 22 MHz and pulsewidth of about 500 ps (FWHM). With the new dye set, no mobility shift correction is required for a separation up to 350 base pairs during separation in a linear 5% PAA gel. This technique of multiplex-dye DNA sequencing shows potential for high-throughput DNA sequencing in parallel capillaries or microfabricated DNA quenching chips.

Diode laser based time-resolved detection and identification of individual mononucleotide molecules in aqueous solution

We applied a short-pulse diode laser emitting at 637 nm with a repetition rate of 30 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual labeled mononucleotide molecules in water. A newly synthesized oxazine dye and the commercially available carbocyanine dye Cy5 were used as fluorescent labels. Multichannel scalar traces, the fluorescence autocorrelation function and fluorescence decay times determined by time- correlated single-photon counting have been measured simultaneously. The time-resolved signals of the two mononucleotides were analyzed and identified by a maximum likelihood estimator. The results showed out that 60 detected photons per transit of a single molecule are sufficient to distinguish two labeled mononucleotides in water with a misclassification of less than 10 percent via their characteristic fluorescence lifetimes of 1.07 +/- 0.27 ns and 1.89 +/- 0.34 ns.

Design of multiplex dyes for the detection of different biomolecules

New fluorescent dyes with characteristic fluorescence lifetimes have been developed for bioanalytical applications. Based on the concept of multiplex dyes, we have designed several rhodamine dyes with nearly identical absorption and emission spectral characteristics but different fluorescence lifetimes. In order to influence the excited state lifetime without changing the spectral characteristics we modified rhodamines with non- conjugated substituents that promote non-radiative transitions. First investigations with covalently coupled biomolecules show the potential of multiplex dyes in DNA sequencing and antigen detection. The biomolecules are identified through the intrinsic fluorescence lifetime of the dye. This principle offers the possibility to make use of different fluorescence lifetimes in each wavelength region. Therefore the number of discernable tags is greatly enhanced. Extending this principle to applications with laser diodes, new rhodamines with functional groups for covalent coupling of analytes have been developed. The new labels exhibit absorption and emission beyond 600 nm and have a high fluorescence quantum efficiency, even in aqueous buffer systems. Time-resolved and intensity measurements of the dyes covalently coupled to a synthetic oligonucleotide are presented. The results obtained in different capillary systems using laser diodes as excitation sources show the potential of these dyes in the red region of the spectrum.

Time-resolved DNA identification in capillary gel electrophoresis with semiconductor lasers

We developed a simple, tiny setup with only a few optical devices for fast and sensitive identification of DNA with a short-pulse semiconductor laser operating at 20 MHz. In combination with newly synthesized fluorescent dyes (rhodamine derivatives) which exhibit high fluorescence quantum efficiencies and distinct fluorescence lifetimes at semiconductor laser excitation wavelength a sensitive detection and time-resolved identification of DNA can be achieved. At an excitation wavelength of 635 nm the fluorescence background is greatly reduced. We demonstrate the DNA identification of A- and G-terminated DNA fragments labeled at the 5-end with the rhodamine derivatives MR 200- 1 and JA 169 during capillary gel electrophoresis. The characteristic time-resolved data are acquired by the time- correlated single-photon counting technique. Time-resolved identification analysis is realized by the maximum likelihood estimator. For prediction of the error rate (misclassification) Cramers equation in combination with a pattern recognition technique is applied. These methods deliver high reliabilities at low classification error rates for low fluorescence light level applications.

Finding the Conformation of Organic Molecules with Genetic Algorithms

Finding the optimal conformation for fluorescent labeled nucleosides is difficult for standard GAs. We investigated the structure of molecule and of the resulting search space itself and concluded that the problem is of the “needle in the haystack” type.

Sensitive detection of p53 antibodies in a homogeneous fluorescence assay format

Circulating p53 autoantibodies are found to be a universal and highly specific tumor marker for malignant diseases. Hence, sereological screening for p53 autoantibodies at low concentration levels has become increasingly relevant for early-stage and follow-up of tumor diagnostics. We developed a new method for the highly sensitive detection of p53 antibodies in a homogeneous fluorescence assay format. Short, linear peptide derived form antibody recognition sequences so human p53 were labeled with an oxazine dye. Hydrophobic interactions constrain a conformation, where the dye interacts selectively with a tryptophan residue in the peptide sequence. Subsequently, the fluorescence of the dye is quenched efficiently due to electron transfer from the indole derivative to the dye in the excited state. Specific antibody recognition induces a conformational change in the peptide structure, repealing the dye-tryptophan interaction. Consequently, a fluorescence increase upon antibody binding signals the binding event. The long-wavelength absorption and emission characteristics of the probe and the use of a red pulsed diode laser as excitation source in a confocal fluorescence microscopic set-up allows ultra sensitive antibody detection at the single-molecule level. The effectiveness of the probes are highlighted by the detection of individual p53 autoantibodies directly in serum dilutions of cancer patients.

Ultrasensitive Detection and Identification of Biomolecules with Diode Lasers - From Dyes to DNA

The increased sensitivity together with the advent of low-cost optical sources and detectors in the visible-near IR region has led us to current efforts to develop new efficient fluorescent labels for biological applications with absorption and emission beyond 600 nm. We applied the pattern recognition technique taken from information theory to ultrasensitive fluorescence detection and identification of dye molecules. Using pulsed diode lasers emitting at 630-640 nm in combination with new efficient rhodamine and oxazine dyes four-dye one-lane DNA sequencing in capillary gelelectrophoresis with time-resolved fluorescence detection is demonstrated.