PRLS_36
Leonid Olga Anatoliy 1, Yevgeniy Povrozin1, Inna Yermolenko1, Iryna Fedyunyayeva 1, and Ewald Terpetschnig2 Yuliya Kudryavtseva
1State
1,2, Patsenker
1, Kolosova
1, Tatarets
Scientific Institution "Institute for Single Crystals", NAS of Ukraine, 60 Lenin Ave., 61001 Kharkov, Ukraine, e-mail: patsenker@isc.kharkov.com, http://www.isc.kharkov.com/old
2SETA
BioMedicals, LLC, 2014 Silver Ct East, Urbana, 61801, IL, USA, e-mail: info@setabiomedicals.com, http://www.setabiomedicals.com
Dyes for Biological Imaging
The higher photostability of Square and SETA probes and labels as compared to Cy or Alexa Fluors is an advantage in biological imaging where longer exposure times help to improve the image quality.
Sprout Mitochondria
pH 5.5
Absorbance 0.08
Fluorescence
Our dye repertoire can be subdivided into fluorescent dyes or luminophores and non-fluorescent dyes called quenchers. Both fluorescent and non-fluorescent dyes are used as reactive labels for covalent attachment to biomolecules, while fluorescent probes undergo non-covalent interactions with biomolecules. Fluorescent dyes are also utilized as classification dyes for single or multiple encoding of reactive microspheres (beads) in suspension arrays.
pH-Sensitive Labels for Biological Applications
These are pH-sensitive markers with tuneable pKa’s in the range from 5.5–10.5:
0.12
1 .0 0 .8 0 .6 0 .4 0 .2 0 .0 5 6 7 8 9 pH 10 11 12 13
Markers for Biomedical Applications
0.04
pH 11.9
Fluorescent Dyes (Luminophores)
Non-fluorescent Dyes (Quenchers)
0.00 400
500 600 W avelength [nm]
700
Classification Dyes for Microspheres
Dye
Reactive Labels
Non-reactive Probes
pH-Sensitive fluorescent labels with pKa’s in the physiological pH range for the investigation of pH-related events in cells and membranes Quenching of fluorescence
Nucleus Human fibroblast cells of conjunctive tissue stained with dye K8-3010 in aqueous media. Plasmatic membrane vesicles are green. Excitation wavelength: 470 nm Human fibroblast cells on a glass slide in 3 days culture stained with dye K83010. Active mitochondria are brightly green while nucleus does not fluoresce. Excitation wavelength: 470 nm
O
NH Biomolecule
Biological species
High Photostability
Dye
1.5 Fluorescence intensity K8-1260 1.0 K8-1643 Alexa-647 0.5 Cy5 0.0
We have developed extremely bright and sensitive fluorescence dyes and real dark quenchers for use in biological and pharmaceutical research, clinical diagnostics, and high-throughput screening (HTS): • Reactive Red and Near-infrared (NIR) Fluorescent Labels of the Square and SETA series for covalent attachment to biomolecules such as proteins, amino-acids, peptides, oligonucleotides, DNA, RNA, lipids and drugs • Fluorescent Probes for proteins, lipids and cells • pH-Sensitive Probes and Labels • Fluorescence Lifetime (FLT) Probes and Labels of SeTau series for FLT and fluorescence polarization based applications • Dark quenchers of the SQ series for Fluorescence Resonance Energy Transfer (FRET) applications • Classification Dyes for single or multiple encoding of microspheres
0
20
40 60 Time [min]
80
100
Normalized fluorescence intensity vs. exposition time for free dyes
Square and SETA dyes of the K8 series are much more photostable compared to Cy5 and Alexa cyanine dyes. This is particularly important in biological imaging applications. For additional information on this topic we refer you to our poster “Thio-squarylium dyes as highly photo-stable markers”
Probes for Fluorescence Lifetime Imaging and Homogeneous Fluorescence Polarization Assays For High- Molecular-Weight Analytes
SeTau tracers show fluorescence in the blue and green spectral region and have FLTs up to 40 ns in water. SeTau dyes are perfectly suited for use in homogeneous fluorescence polarization assay of high molecular-weight antigens and substantial polarization increases are observed upon binding of the high molecular-weight tracers to the antibody (see below). Red and near-IR microenvironment-sensitive probes exhibit lifetime increases from 500 ps up to 2–3 ns upon binding to biomolecules. These probes have high affinity for proteins, biomembranes and lipoproteins and are useful to detect and quantitate these analytes.
350 300 250 200 150 0.0 K7-545 — BSA + nonspecific IgG 0.2 0.4 0.6 0.8 1.0 1.2 [ANTI-BSA / K7-545-BSA] 1.4 K7-545 — BSA + anti-BSA
Human fibroblast cells stained with potential-sensitive dye K5-1000. Nucleus is orange, plasma membrane vesicles are orange-brown. Excitation wavelength: 470 nm
Human fibroblast cells stained with dye K8-1355. Plasma membrane vesicles are orange-red. Excitation wavelength: 580 nm
Wide Spectral Range
These dyes, probes and labels have several advantages as compared to other commercially available probes and labels: Square and SETA dyes absorb (300–850 nm) and emit (500–850 nm) in a wide spectral range. Unlike dyes of the Cy and Alexa Fluor series, these red and NIR emitting markers can be excited not only with the red, 635-nm and 670-nm diode lasers but also with the UV and blue diode lasers and light emitting diodes (LEDs) at 380-nm, 405-nm and 436-nm.
1.0
Human fibroblast cells stained with K8-1400. Nucleus is yellow-orange, plasma membrane vesicles are red. Excitation wavelength: 580 nm Normal cell
Human fibroblast cells stained with K81500. Nucleus is yellow-orange, plasma membrane vesicles are red. Excitation wavelength: 580 nm
0.6 0.4 0.2 0.0 300 400 500 600 700 800
Polarization [mP]
0.8 Absorption
Wavelength [nm]
Changes in Fluorescence Polarization of K7-545-labeled BSA (MW ~ 65 kDa) upon titration with anti-BSA. The labeled BSA species has still a relatively low polarization of 165 mP and only upon addition of specific antibody the polarization increases gradually to its final value of 335 mP, which demonstrates the usefulness of this label for the measurement of high-molecular-weight antigens in a Fluorescence Polarization Immunoassay (FPA)
Necrotic cell with disrupted wall
Mitochondria (yellow)
Brightness
70 60 Quantum Yield [%] 50 40 30 20 10 0 0 1 Alexa-647 — BSA conjugates Cy5 — BSA conjugates 2 3 Dye-to-Protein Ratio 4 K8-1643 — BSA conjugates
Dark Quenchers
Our reactive Super Quenchers of the SQ series absorb in the 500– 800 nm spectral range. They exhibit no residual fluorescence and are perfectly suited for covalent labeling of proteins, peptides and oligo-nucleotides for use in FRET and real-time PCR based applications.
Number K8-1649 K8-2602 Product Name SQ740-mono-NHS SQ755-di-NHS Absorption max. [nm] 740 698, 756 Extinction Coefficient [M–1cm–1] 44,000 200,000 Solubility Water, Ethanol, DMF, DMSO Water, Ethanol, DMF, DMSO
Square and SETA dyes of the K8 series have high extinction coefficients (up to 265,000 M–1cm–1). Protein conjugates of these dyes are extremely bright. Quantum yields (up to 70%) for our conjugates are unmatched by Cy5 or Alexa dyes.
Yeast cells (Saccharomyces cerevisiae) stained with dye K8-3010. Cell walls are bright green. Necrotic cells with disrupted cell walls have bright green fluorescence from cell interior. Excitation wavelength: 470 nm
Yeast cells (Saccharomyces cerevisiae) stained with dye K8-1400. Nucleus does not fluoresce, cytoplasm is red, active mitochondria are yellow. Excitation wavelength: 580 nm
Classification Dyes for Microspheres
for a wide spectral range
Microspheres stained with classification dyes
2–20 µm
Linker
Sensitivity to Microenvironment
150 Square-670 +10 Fluorescence 100 Square-670 free in solution
–4
Reactive group
Absorbance [a.u.]
M BSA
50
0
650
700 W avelength [nm ]
750
Red and near-IR probes and labels show noticeable increases of fluorescence intensity and longer fluorescence lifetimes in presence of large biomolecules such as proteins and lipids.
1.0 0.8 0.6 0.4 0.2 0.0 300 400 500 600 700 800 900 1000 Wavelenght [nm] No Fluorescence
Read-out color code
Read-out positive event
Bioanalyte
Reference Dye
The work was supported by the STCU grants No. U111, P313, 3795, and 3804