Studies of monomeric and homodimeric oxazolo[4,5-b]pyridinium cyanine dyes as fluorescent probes for nucleic acids visualization.

The series of recently synthesized monomeric and homodimeric cyanine dyes based on monomethine cyanine chromophore with oxazolo[4,5-b]pyridinium and quinoline end groups [Vassilev A, Deligeorgiev T, Gadjev N, Drexhage K-H. Synthesis of novel monomeric and homodimeric cyanine dyes based on oxazolo[4,5-b]pyridinium and quinolinium end groups for nucleic acid detection, Dyes Pigm 2005;66:135-142] were studied as possible fluorescent probes for nucleic acids detection. Significant fluorescence enhancement and intensity level (quantum yield up to 0.75) was observed for all the dyes in the presence of DNA. The oxazolo[4,5-b]pyridinium cyanines demonstrated high sensitivity as fluorescent stains for post-electrophoretic visualization of nucleic acids in agarose gels upon both VIS and UV transillumination, and the visualized band contained 0.8 ng of dsDNA.

New fluorescent markers for the red region.

Two new classes of fluorescent dyes have been developed as labels for the red region of the spectrum: amide-bridged benzopyrylium dyes and carbopyronin dyes. The fluorescence quantum yield ranges from 20 to 90%, the decay time from 1 to 4 ns. The pH- and solvent-dependence of absorption and fluorescence are described in detail. Covalent attachment is possible via activated carboxyl groups.

Homodimeric monomethine cyanine dyes as fluorescent probes of biopolymers.

The fluorescence properties of newly synthesized homodimeric monomethine cyanine dyes in the presence of biopolymers are investigated. They do not fluoresce in TE buffer and bidistilled water but become strongly fluorescent (Q(F)=0.3-0.9) in the region 530-650 nm when bound to dsDNA and ssDNA. The detection limit of dsDNA is about 1.7 ng/ml. Some of dyes studied are able to distinguish between dsDNA and ssDNA, RNA, BSA in solution and gel electrophoresis. The influence of different factors (temperature, pH and viscosity of the medium, presence of histone) on the formation of the dye-biopolymer complexes is investigated. The results of steady-state and dynamic fluorescence measurements concerning the different types of binding between dyes and biopolymers show that the new dyes are applicable in molecular biology as highly sensitive fluorescence labels.

Multiplex dye DNA sequencing in capillary gel electrophoresis by diode laser-based time-resolved fluorescence detection.

A new one-lane, four-dye DNA sequencing method was developed which is based on time-resolved detection and identification of fluorescently labeled primers. For fluorescent labels, we used two newly synthesized rhodamine derivatives (MR200-1, JA169), a new oxazine derivative (JA242), and a commercially available cyanine dye (CY5). The dye fluorescence was excited by a pulsed diode laser emitting at 630 nm. The fluorescence decay was detected by an avalanche photodiode using a single-filter system. The dyes used here, so-called multiplex dyes, can be distinguished and identified via their fluorescence decay patterns. The DNA fragments were labeled at the primer using linkers of various lengths and positions. For separation of the enzymatically generated DNA fragments, capillary gel electrophoresis (CGE) with a 5% linear polyacrylamide gel was employed. On covalent attachment to oligonucleotides, the dyes exhibit fluorescence decay times of 3.7 (MR200-1), 2.9 (JA169), 2.4 (JA242), and 1.6 ns (CY5) measured during CGE. The CGE mobility of the labeled DNA fragments could be controlled and nearly equalized by the coupling position and the linker length. First, time-resolved, one-lane, four-dye DNA sequencing runs in CGE are presented. The sequence information of 660 bp was determined with a probability of correct classification of > 90%. This result was obtained directly from the raw data without any of the mobility corrections that are necessary with other methods.

Sequence verification by hybridisation with fluorescent octanucleotides as a first step to a fluorescent sequencing by hybridisation protocol.

Three sets of partly overlapping octanucleotides are 5' labelled with derivates of the fluorescence dyes fluorescein-, coumarine- and rhodamine, respectively. Hybridisation conditions are determined, under which all octanucleotides hybridise correctly against complementary target sequences bound on nylon membranes. Target sequences are three synthetic 48-mer oligonucleotides and herring sperm DNA, a positive control containing almost all possible octanucleotides. None of the octanucleotides hybridised to incorrect target sequences. Analysing these results, a given sequence could be unambiguously verified. A feature critical for the accuracy of the hybridisation is the temperature during the last washing step. This temperature can be estimated using the equation T = 19 - 0.4(G + C) + 0.15(G + C)2. Using octanucleotides labelled with three different colors, three hybridisations can be performed simultaneously.

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.

Simultaneous antigen detection using multiplex dyes.

To detect several antigens simultaneously, antibodies directed against different antigens were immobilized on a quartz surface. The antigens were tagged with multiplex, dyes, which show different fluorescence lifetimes but similar excitation and emission spectra. The antigens were detected by recognizing the characteristic fluorescence lifetime. Furthermore, the effect of labeling of the dye on the antigen molecules was examined.

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.

Compact arc lamp of high luminance.

A continuous high pressure arc lamp operating with argon is described in detail. Owing to a special electrode design a very high brightness of 160 W/(nm-sr-cm(2)) at an electrical input of 5.3 kW is reached near the cathode.

Continuous-wave dye laser pumped by a high-pressure argon arc.

Continuous-wave operation of a Rhodamine 6G dye laser, incoherently pumped by a high-pressure argon arc, has been achieved. A special electrode design reduces melting of the electrode tips, and thus the arc provides the necessary brightness for periods of the order of hours.

Near-infrared dye having a large ultrafast third-order susceptibility.

Efficient degenerate four-wave mixing of 160-psec infrared light pulses at lambda = 1.064 microm in solutions of a new dye that absorbs between 1.0 and 1.6 microm is reported. A Kerr-like third-order susceptibility of X((3))(xyyx) = 8 +/- 3) x 10(-12) esu with a decay time of less than 50 psec is observed in various solvents.

Tunable picosecond pulses around 1.3 microm generated by a synchronously pumped infrared dye laser.

An infrared dye laser is synchronously pumped by a cw mode-locked Nd:YAG system at 1.06 microm. The tuning range extends from 1.20 to 1.32 microm. A pulse duration of 3 psec was measured at 1.25 microm. The high photostability of the laser dye makes the laser system a practical device.