New fluorescent dyes in the red region for biodiagnostics (original) (raw)
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Red-Emitting Rhodamine Dyes for Fluorescence Microscopy and Nanoscopy
Chemistry - A European Journal, 2010
Owing to their high photostabilities, high extinction, high fluorescent quantum yields, and low degree of triplet formation, rhodamine dyes are widely used as laser dyes and fluorescent markers for labeling proteins, nucleic acids, lipids, carbohydrates, toxins, hormones, and other biomolecules. [1] Besides their use in biological imaging and single-moleculebased spectroscopy, rhodamines served well in the practical implementation of some new physical concepts, for example, those that helped to overcome the diffraction limit in (far-field) optical microscopy. "Nanoscopic" techniques [2] such as STED (stimulated emission depletion), [2a, d, h] PALM (photoactivation localization microscopy), [2i, s] STORM (stochastic reconstruction microscopy), [2j, t-v] and GSDIM (ground-state depletion with individual molecular return) [3] allowed the optical resolution to be improved from about 200-350 nm to 20-35 nm by switching between the dark and the bright states of a fluorescent marker. However, the rhodamine family lacks water-soluble compounds that combine high photostabilities and fluorescence quantum yields with absorbance and fluorescence band maxima lying in the far-red optical region.
Chemistry - A European Journal, 2012
Fluorescent dyes emitting red light are frequently used in conventional and super-resolution microscopy of biological samples, although the variety of the useful dyes is limited. We describe the synthesis of rhodamine-based fluorescent dyes with absorption and emission maxima in the range of 621-637 and 644-660 nm, respectively and demonstrate their high performance in confocal and stimulated emission depletion (STED) microscopy. New dyes were prepared by means of reliable chemical transformations applied to a rhodamine scaffold with three variable positions. They feature polarity, water solubility, variable net charges, improved stabilities of N-hydroxysuccinimidyl (NHS) esters, as well as large fluorescence quantum yields in dye solutions and antibody conjugates. The photophysical and imaging properties of dyes containing three different polar groups, namely primary phosphate, sulfonic acid (in two different positions), and hydroxyl were compared. A dye with two primary phosphate groups was explored as a valuable alternative to dyes with "classical" sulfonic acid groups. Due to the increased net charge of the phosphorylated dye (q=-4 at pH 8), it demonstrated a far better electrophoretic mobility compared with analogues with two sulfonic acid groups (q=-2). As an example, one fluorescent dye was designed to be especially convenient for practical use. It is characterized by sufficiently high chemical stability of the NHS ester, its simple isolation, handling, and solubility in aqueous buffers, as well as in organic solvents. All these features, accompanied by a zero net charge in conjugates, were accomplished by the introduction of hydrophilic groups of two types: two hydroxyl groups and one sulfonic acid residue.
Fluorescent Probes and Labels for Biomedical Applications
Annals of the New York Academy of Sciences, 2008
Fluorescence probes and labels have become indispensable tools for clinical diagnostics, highthroughput screening, and other biomedical applications. We have developed several classes of new squaraine-based red and near-infrared (NIR) probes and labels (SETA and Square series), naphthalimide-based fluorescence lifetime dyes (SeTau series), and cyanine-and squaraine-based quenchers (SQ series). This report discusses the spectral and photophysical properties of these new markers. In particular, the red and NIR dyes of the SETA and Square series are extremely bright, with photostabilities that are unmatched by any other dyes in the same spectral region.
Noncovalent Labeling of Biomolecules with Red and Near- Infrared Dyes
Molecules, 2004
Biopolymers such as proteins and nucleic acids can be labeled with a fluorescent marker to allow for their detection. Covalent labeling is achieved by the reaction of an appropriately functionalized dye marker with a reactive group on a biomolecule. The recent trend, however, is the use of noncovalent labeling that results from strong hydrophobic and/or ionic interactions between the marker and biomolecule of interest. The main advantage of noncovalent labeling is that it affects the functional activity of the biomolecule to a lesser extent. The applications of luminescent cyanine and squarylium dyes are reviewed.
New Fluorescence Probes for Biomolecules
Molecules, 2015
Steady state fluorescence measurements have been used for the investigation of interaction between the bovine serum albumin (BSA) and fluorescence probes: 3-hydroxy-2,4bis[(3-methyl-1,3-benzoxazol-2(3H)-ylidene)methyl]cyclobut-2-en-1-one (SQ6), 3-hydroxy-2,4-bis[(3-methyl-1,3-benzothiazol-2(3H)-ylidene)methyl]cyclobut-2-en-1-one (SQ7) and 3-hydroxy-2,4-bis[(1,3,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene)methyl]cyclobut-2-en-1-one (SQ8). The binding constant between bovine serum albumin and squarine dyes has been determined by using both the Benesi-Hildebrand and Stern-Volmer equations. The negative value of free energy change indicates the existence of a spontaneous complexation process of BSA with squarine dyes.
Journal of Histochemistry & Cytochemistry, 2003
Transplantation and Immunology Branch, NCI-NIH, Bethesda, Maryland (WGT) S U M M A R Y Amine-reactive N -hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N -hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications. (
Chemistry - A European Journal, 2014
The synthesis, reactivity, and photophysical properties of new rhodamines with intense red fluorescence, two polar residues (hydroxyls, primary phosphates, or sulfonic acid groups), and improved hydrolytic stability of the aminoreactive sites (NHS esters or mixed N-succinimidyl carbonates) are reported. All fluorophores contain an N-alkyl-1,2-dihydro-2,2,4-trimethylquinoline fragment, and most of them bear a fully substituted tetrafluoro phenyl ring with a secondary carboxamide group. The absorption and emission maxima in water are in the range of 635-639 and 655-659 nm, respectively. A vastly simplified approach to redemitting rhodamines with two phosphate groups that are compatible with diverse functional linkers was developed.
Bioconjugate Chemistry, 2013
The rational design of bright optical probes and dye-biomolecule conjugates in the NIRregion requires fluorescent labels that retain their high fluorescence quantum yields when bound to a recognition unit and/or upon interaction with a target. As hydrophilicity-controlled dye aggregation in conjunction with homo-FRET presents one of the major fluorescence deactivation pathways in dye-protein conjugates, fluorescent labels are required that enable higher labeling degrees with minimum dye aggregation. Aiming at a better understanding of the factors governing dye-dye interactions, we systematically studied the signal-relevant spectroscopic properties, hydrophilicity, and aggregation behavior of the novel xS-IDCC series of symmetric pentamethines equipped with 2, 4, and 6 sulfonic acid groups and selected conjugates of these dyes with IgG and the antibody cetuximab (ctx) directed against the Page 1 of 41 ACS Paragon Plus Environment Bioconjugate Chemistry 2 cancer-related epidermal growth factor (EGF) receptor in comparison to the gold standard Cy5.5. With 6S-IDCC that displays a molar absorption coefficient of 190,000 M-1 cm-1 and a fluorescence quantum yield (Φ f) of 0.18 in aqueous media like PBS and nearly no aggregation, we could identify a fluorophore with a similarly good performance as Cy5.5. Bioconjugation of 6S-IDCC and Cy5.5 yielded highly emissive targeted probes with comparable Φ f values of 0.29 for a dye-to-protein (D/P) ratio < 1 and a reduced number of protein-bound dye aggregates in the case of 6S-IDCC. Binding studies of the ctx-conjugates of both dyes performed by fluorescence microscopy and FACS revealed that the binding strength between the targeted probes and the EGF receptor at the cell membrane is independent of D/P ratio. These results underline the importance of an application-specific tuning of dye hydrophilicity for the design of bright fluorescent reporters and efficient optical probes. Moreover, we could demonstrate the potential of fluorescence spectroscopy to predict the size of fluorescence signals resulting for other fluorescence techniques such as FACS. cyanine / cetuximab / IgG / protein labeling / fluorescence quantum yield / hydrophilicity / dimerization constant
An in vitro characterization study of new near infrared dyes for molecular imaging
European Journal of Medicinal Chemistry, 2009
The spectroscopic properties, stability, and cytotoxicity of series of cyanine labels, the dyes DY-681, DY-731, DY-751, and DY-776, were studied to identify new tools for in vivo fluorescence imaging and to find substitutes for DY-676 recently used by us as fluorescent label in a target-specific probe directed against carcinoembryonic antigen (CEA). This probe enables the selective monitoring of CEA-expressing tumor cells in mice, yet displays only a low fluorescence quantum yield and thus, a non-optimum sensitivity. All the DY dyes revealed enhanced fluorescence quantum yields, a superior stability, and a lower cytotoxicity in comparison to clinically approved indocyanine green (ICG). With DY-681 and far-red excitable DY-731 and DY-751, we identified three dyes with improved properties compared to DY-676 and ICG.