Fluorescence squaraines

Ioffe VM, Gorbenko GP, Domanov YA, Tatarets AL, Patsenker LD, Terpetsching EA, Dyubko TS (2006) A new fluorescent squaraine probe for the measurement of membrane polarity. J Fluoresc 16 47-52... 

The fluorescent squaraine labels Seta-670-mono-NHS and Seta-635-NH-... 

Figure 22 Optical ISC approach anion-mediated inhibition of the reaction of a colored and fluorescent squaraine dye to its leuko form. 2...

The sulfo-gmup containing squaraine-taurine probe 8 [62] displayed a very high affinity for BSA and other blood proteins. This probe exhibits low quantum yields and short fluorescence lifetimes in water and a significant increase of these characteristics upon binding to proteins. 

Hydrophobic (10a) and hydrophilic (10b) squaraines show a noticeable increase in fluorescence intensity in presence of HSA and importantly dye 10b, containing a sulfo group, exhibits a large intensity increase when bound to avidin, a protein well-known to quench many fluorescent dyes [58]. 

The quantum yields are 0.15-0.21 in ethanol and 0.01-0.02 in an aqueous medium, but in micelles, the quantum yields are five to tenfold increased. The aggregation of these dyes was studied in [53]. The amphiphilic squaraines 4 combine favorable photophysical properties and good solubility in aqueous media and in addition interact efficiently with micelles, and therefore have the potential to be used as NIR fluorescent sensors. However, our own investigations show that aniline-based squaraines lack chemical and photochemical stability when compared to oxo-squaraines with heterocyclic end-groups. 

Dialkylanthracene-containing squaraine dyes 17 show intense absorption and emission in the NIR region (720-810 nm) [74]. They are compatible with aqueous environments and show substantial enhancement of quantum yields and fluorescence lifetimes in hydrophobic and micellar media, suggesting that these dyes can be potentially useful as fluorescent probes in biological applications, e.g., for imaging of hydrophobic domains such as cell membranes. 

Also, the recently introduced hydrazone end-capped squaraines 19 [77], absorbing in the 600-800-nm range, could be promising candidates for the development of fluorescent probes. 

An r/rr/mnv -squaraine-based chemosensor 23a absorbing at 635 nm (eM = 260,000 M 1cm 1) and emitting at 665 nm signals alkaline and earth-alkaline metal ions in millimolar concentrations in acetonitrile [81]. In presence of Na+ ions, the fluorescence signal weakly increases while it significantly decreases in presence of Ca2+, Mg2+, and Ba2+ and does not change substantially upon addition of K+ ions. The same squaraine 23a and the azacrown-squaraine 23b [82] were used for Na+ and K+ sensing in a plasticized PVC matrix [83]. The squaraine derivatives exhibited fluorescence emission based optical responses to... 

A long-wavelength probe 29 signaling carbohydrates in aqueous solutions by increasing of fluorescence was developed by Akkaya and Kukre on the basis of a symmetrical squaraine dye containing two phenylboronic acid functions [89]. The emission maximum of this probe is at 645 nm. A maximal response of about 25% was found for fructose. 

The dihydroxyaniline-squaraine chromophore was used by Akkaya and Isgor in the fluorescent chemosensor 30 for the measurement of pH [90]. This chemosensor, having the molar absorptivity about 200,000 M em 1 and quantum yield 0.2,... 

Because squaraines are sensitive toward nucleophilic attack resulting in decoloration and fluorescence quenching, they can be used as specific chemodosimeters for t/i/oZ-containing compounds. Squaraines 4 have been successfully applied as sensors for low-molecular-weight aminothiols like cysteine in a complex multicomponent mixture (e.g., human plasma) [91]. 

Fluorescent labels based on oxo-squaraines were described in numerous articles and patents [45, 52]. Mono-reactive hydrophobic (32a) and hydrophilic (32b) squaraine labels containing one NHS ester group were synthesized by Terpetschnig et al. [62]. The initially low quantum yields and short fluorescence lifetime of 32b in aqueous solutions significantly increase after covalent binding to proteins. 

The NIR fluorescent 2,3-dihydroperimidine-squaraines 35a,b were synthesized and used as biological labels [99]. Dye 35b including four sulfopropyl groups exhibits high water-solubility, an emission maximum at 812 nm (protein ratio D/P = 1), and 817 nm in the presence of BSA, which indicates compatibility with commercially available NIR laser diodes. 

A thiol-reactive squaraine 36 (iodoacetamide) that displays fluorescence emission above 650 nm was used to develop a reagentless glucose monitoring assay [102]. 

Squaraine dyes 10b, 39a, 39b, 41a, 41c, 41d, and 41e were used to measure different proteins such as BSA, HSA, ovalbumin, avidin from hen egg white, lysozyme, and trypsin (Fig. 12) [58]. It is difficult to predict correlations between the dyes structures and the affinity or sensitivity of the dyes for different proteins. All squaraine probes exhibit considerable fluorescence increases in the presence of BSA. Dicyanomethylene-squaraine 41c is the brightest fluorescent probe and demonstrates the most pronounced intensity increase (up to 190 times) in presence of BSA. At the same time, the fluorescent response of the dyes 10b, 39a, 39b, 41a, 41c, 41d, and 41e in presence of other albumins (HSA and ovalbumin) is, in general, significantly lower (intensity increases up to 24 times). Dicyanomethylene-squaraine 41a and amino-squaraines 39a and 39b are the most sensitive probes for ovalbumin. Dyes 41d, 10b, and 41e containing an A-carboxyalky I -group demonstrate sufficient enhancement (up to 16 times) in the presence of avidin. Nevertheless, the presence of hydrolases like lysozyme or trypsin has only minor effects on the fluorescence intensity of squaraine dyes. 

The dicyanomethylene-squaraine dye 41e was found to be highly sensitive to trace protein-lipid interactions [109]. Lysozyme association with the lipid bilayer leads to a noticeable decrease in the fluorescence intensity of 41e. In a separate... 

Ring-substituted squaraines such as 42 and 43 [98, 111] also can be used as fluorescent probes however, the quantum yields for d/t/no-squaraines 42 are lower compared to monothio-squaraines or aro-squaraines. 

Compared to oxo-squaraines or other ring-substituted squaraines, amino-squaraines 39 [45, 52, 112] have ionic character, similar to open-chain cyanine dyes, and due to the positive net charge, these dyes are to some extend water-soluble. Amino-squaraines absorb and emit at longer wavelength than the corresponding oxo-squaraines the absorption maxima are between 650-710 nm (eM = 85,000-300,000 M-1cm-1) [45, 112], The increase in solvent polarity is accompanied by a hypsochromic shift of the absorption. Amino-squaraine dyes are potentially used as fluorescent probes but because their photostability is inferior to those of oxo-squaraines and other ring substituted squaraines of similar structure, their applications are rather limited. 

The commercially available dicyanomethylene squaraine dye Seta-670-mono-NHS showed extremely low blinking effects and good photostability when used in single-molecule studies of multiple-fluorophore labeled antibodies [113]. Seta-670-mono-NHS and Seta-635-NH-mono-NHS were covalently labeled to antibodies and used in a surface-enhanced immunoassay [114]. From the fluorescence intensity and lifetime changes determined for a surface that had been coated with silver nanoparticles, both labeled compounds exhibited a 15- to 20-fold... 

While open-chain cyanines such as Cy5, Cy5.5, or Alexa 647 typically exhibit small fluorescence lifetime changes upon binding to biomolecules, squaraine dyes, in general, demonstrate pronounced lifetime changes upon binding to large-molecular-weight analytes [17, 18], which can be used for FLT-based assays. 

Recently, SETA BioMedicals has developed a new near-infrared squaraine-based label Seta-633, which can be used to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter [19]. This label exhibits lower quantum yields and shorter fluorescence lifetimes when free in solution, but these values substantially increase upon interaction with proteins, which is contrary to tracers like Cy5 or Alexa 647. It was demonstrated in a model assay that a biotinylated Seta-633 binds to anti-biotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 2.76 fold upon binding to a specific antibody (anti-biotin, MW =160 kDa), while the titration with BSA or nonspecific antibody does not result in a noticeable change in lifetime (Fig. 13). The label is compatible with readily available light sources (635 nm or 640 nm lasers) and filter sets (as for Cy5 or Alexa 647) and its... 

Cyanine and squaraine dyes with hydrogen substituents on the indolenine-nitrogen in one or both of the heterocyclic end-groups, the so-called norcyanines and norsquaraines, are useful as fluorescent pH-indicators due to the reversible equilibrium between their protonated and deprotonated forms ... 

Due to their low sensitivity toward the environment, cyanine dyes are perfect candidates as fluorescent labels. Squaraine dyes on the other hand display a highly environment-sensitive response and are therefore not only useful as fluorescent probes and labels but also, in particular, well-suited for lifetime-based applications. 

Due to their displayed higher photostability, indolenine-based cyanines and squaraines are preferred for the design of fluorescent tracers. Dicyanomethylene- and thio-squaraines with substituted squaraine oxygens also show potential as highly photostable fluorescent probes. 

Tatarets AL, Fedyunyayeva IA, Dyubko TS, Povrozin YA, Doroshenko AO, Terpetschnig EA, Patsenker LD (2006) Ring-substituted squaraine dyes as probes and labels for fluorescence assays. Anal Chim Acta 570 214-223... 

Reddington MV (2007) Synthesis and properties of phosphonic acid containing cyanine and squaraine dyes for use as fluorescent labels. Bioconjugate Chem 18 2178-2190... 

Volkova KD, Kovalska VB, Tatarets AL, Patsenker LD, Kryvorotenko DV, Yarmoluk SM (2007) Spectroscopic study of squaraines as protein-sensitive fluorescent dyes. Dyes Pigm... 

Terpetschnig E, Szmacinski H, Ozinskas A, Lakowicz JR (1994) Synthesis of squaraine-iV-hydroxysuccinimide esters and their biological application as long-wavelength fluorescent labels. Anal Biochem 217 197-204... 

Arun KT, Ramaiah D (2005) Near-infrared fluorescent probes synthesis and spectroscopic investigations of a few amphiphilic squaraine dyes. J Phys Chem A 109 5571-5578... 

Basheer MC, Santhosh U, Alex S, Thomas KG, Suresh CH, Das S (2007) Design and synthesis of squaraine based near infrared fluorescent probes. Tetrahedron 63 1617-1623... 

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