Kyung-Tae Han

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.

SINGLE-MOLECULE COUNTING AND IDENTIFICATION IN A MICROCAPILLARY

Abstract Using a confocal microscope we studied photon bursts from individual molecules (dye-labeled mononucleotides) flowing in a cone-shaped microcapillary with an inner diameter of 0.5 μm at the small end of the cone. The flow of the conjugates was established by electrokinetic forces. Excitation of the fluorophore was provided by a pulsed diode laser (λ=640 nm, average power 800 μW, repetition rate 56 MHz). The characteristic diffusion and flow time through the laser focus and burst size statistics were determined in the microcapillary as well as in an open volume. Applying time-correlated single-photon counting, two different conjugate species (Cy5-dCTP, JA53-dUTP) can be distinguished due to their characteristic fluorescence decay time with a probability of correct classification of 80%.

Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level

We report on confocal fluorescence lifetime imaging microscopy (CFLIM) of single dye molecules adsorbed on glass surface. Applying a short-pulse diode laser emitting at 635 nm with a repetition rate of 64 MHz we studied the time-resolved identification of individual carbocyanine and oxazine dyes via their characteristic fluorescence lifetimes of 2.06±0.37 ns (Cy5) and 3.89±0.91 ns (JA242). Fluctuations in fluorescence intensity and lifetime of individual adsorbed molecules were investigated with millisecond time resolution. These jumps exhibit short off-states τoff of 0.5 ms which can be ascribed to the triplet state lifetime under dry conditions with an intersystem crossing yield YISC of ∼0.2 %. Besides triplet states, other quantum jumps into longer lived states (several milliseconds) with lower transition probability were observed. The correlation of rotational and spectral jumps of single and coupled fluorophores with changes in the observed fluorescence lifetime are discussed.

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.

Sensitive fluorescence detection in capillary electrophoresis using laser diodes and multiplex dyes

Abstract A detector for laser induced fluorescence based on laser diole technology was designed and sensitivity measurements with rhodamine 800 were performed. The detection limit is around 100 molecules in the detection volume. Using the laser diode in combination with a commercial capillary electrophoresis system, about 1200 molecules were detectable. Using time-resolved fluorescence measurements multiplex dyes were recognized with a pulsed laser diode and a pattern recognition technique collecting only a few hundred photons.

Sensitive fluorescence detection using laser diodes and multiplex dyes

Abstract A laser diode based detector for laser-induced fluorescence was designed. Sensitivity measurements with rhodamine 800 showed a detection limit of 100 molecules. Using time-resolved fluorescence measurements new fluorescent dyes, called multiplex dyes, were recognized with a pulsed laser diode by their characteristic fluorescence lifetime and a pattern recognition technique collecting only a few hundred photons.

Biodiagnostics with multiplex dyes

A new bioanalytical concept that uses the characteristic fluorescence lifetimes of new multiplex dyes as an identification parameter is presented. Several dyes can be distinguished at the same wavelength by recognition of the fluorescence lifetimes. It is shown that the identification can be achieved in a capillary gel electrophoresis system in a few milliseconds with a misclassification rate of 10-4. As an example the simultaneous detection of two antibodies at one wavelength is presented.