Pyrene Excimer Research Articles

Fluorescent probes for nucleic Acid visualization in fixed and live cells.

This review analyses the literature concerning non-fluorescent and fluorescent probes for nucleic acid imaging in fixed and living cells from the point of view of their suitability for imaging intracellular native RNA and DNA. Attention is mainly paid to fluorescent probes for fluorescence microscopy imaging. Requirements for the target-binding part and the fluorophore making up the probe are formulated. In the case of native double-stranded DNA, structure-specific and sequence-specific probes are discussed. Among the latest, three classes of dsDNA-targeting molecules are described: (i) sequence-specific peptides and proteins; (ii) triplex-forming oligonucleotides and (iii) polyamide oligo(N-methylpyrrole/N-methylimidazole) minor groove binders. Polyamides seem to be the most promising targeting agents for fluorescent probe design, however, some technical problems remain to be solved, such as the relatively low sequence specificity and the high background fluorescence inside the cells. Several examples of fluorescent probe applications for DNA imaging in fixed and living cells are cited. In the case of intracellular RNA, only modified oligonucleotides can provide such sequence-specific imaging. Several approaches for designing fluorescent probes are considered: linear fluorescent probes based on modified oligonucleotide analogs, molecular beacons, binary fluorescent probes and template-directed reactions with fluorescence probe formation, FRET donor-acceptor pairs, pyrene excimers, aptamers and others. The suitability of all these methods for living cell applications is discussed.

Effect of Side-Chain Length on the Polymer Chain Dynamics of Poly(alkyl methacrylate)s in Solution

Eight series of poly(alkyl methacrylate)s bearing different side chains and one series of poly(methyl acrylate) were randomly labeled with pyrene, and their ability to form pyrene excimer was characterized quantitatively by steady-state and time-resolved fluorescence to demonstrate that such measurements provide a quantitative measure of polymer chain dynamics (PCD) in solution. Each series of pyrene-labeled polymer showed increased excimer formation with increasing pyrene content, but the increase was more pronounced for the polymers known to be more flexible based on their reported glass transition temperature (Tg). In the case of the poly(alkyl methacrylate)s with a linear side chain, a shorter side chain resulted in increased excimer formation. Replacing a linear side chain with a more rigid one containing the same number of carbon atoms resulted in decreased mobility of the polymer. Fluorescence Blob Model (FBM) analysis of the fluorescence decays provided a more accurate representation of those pyrenes that formed excimer by diffusion and thus reflected PCD more precisely. Global FBM analysis of the pyrene monomer and excimer fluorescence decays yielded the blob size Nblob and the product kblob × Nblob which reflects PCD. For each series, both Nblob and kblob × Nblob remained constant with pyrene content. Their average value ⟨Nblob⟩ and ⟨kblob × Nblob⟩ decreased substantially with increasing side-chain length, addition of the α-methyl substituent to poly(methyl acrylate) to yield poly(methyl methacrylate), or increased rigidity of the side chain, demonstrating that an increase in bulkiness or stiffening of the side- or main chain is associated with a pronounced decrease in chain mobility. These experiments are the first to demonstrate that pyrene excimer formation can be used to characterize quantitatively PCD in solution in the same manner that Tg is being used to characterize PCD in the bulk.

Membrane damages under high pressure of human erythrocytes agglutinated by concanavalin A

Human erythrocytes are agglutinated by lectins such as concanavalin A (Con A). The behaviors of agglutinated erythrocytes under pressure are less well understood. Here, we report the effects of erythrocyte agglutination on pressure-induced membrane damages. Small clumps of intact erythrocytes by Con A were dissociated by a pressure of 200MPa. Further, the observation by scanning electron microscopy demonstrated the generation of vesicles, fragmented particles, and membrane hole. On the other hand, large clumps of trypsin-digested erythrocytes by Con A seemed to be stable against 200MPa. However, the erythrocytes dissociated from such pressure-treated clumps by methyl α-mannopyranoside also showed the existence of vesicles and fragmented particles except for the membrane hole. Pressure-induced hemolysis was greatly suppressed in such large clumps. Similar suppressive effects were observed in erythrocytes packed by centrifugation. However, the hemolysis occurred when the erythrocytes dissociated from 200MPa-treated large clumps by methyl α-mannopyranoside were incubated at 0°C and atmospheric pressure. Pyrene excimer fluorescence due to spectrin denaturation was observed in Con A-agglutinated ghosts that were exposed to a pressure of 200MPa. These results suggest that upon pressure treatment of tightly agglutinated erythrocytes, the hemolysis is greatly suppressed, but membrane damages occur such as spectrin denaturation and vesiculation.

Study on fluorescent switching of naphthopyran and pyrene-containing dyad and copolymer in solutions and films

The fluorescent photo-switching systems were prepared based on fluorescent pyrene and photochromic naphthopyran in the forms of organic dyad and copolymer. Naphthopyran in two systems displayed excellent photochromic performance, especially in tetrahydrofuran solution. The fluorescent emission was modulated between “on” and “off” via the photoisomerization of naphthopyran in high-degree, especially in polymer (>90%) due to the photoinduced energy transfer from pyrene excimers to the open-form naphthopyran. Both the fluorescence photoswitching and the photochromism of polymer in solution displayed excellent fatigue resistance. The fluorescent image shows the patterned irradiation of the polymer-doped polymethylmethacrylate film can be recorded by fluorescent pattern on the sample. The non-destructive readout ability of polymer-doped polymethylmethacrylate film was achieved.

Mechanically Induced Multicolor Switching Based on a Single Organic Molecule

Asserting its independence: A single organic molecule containing pyrene and rhodamine B chromophores separated by a peptide spacer formed a mechanochromic material whose fluorescence could be switched from blue to bluish-green and reddish by grinding. Modulation of the overlapped packing pattern of the pyrene excimer for blue emission was crucial for the reversible tricolored switching observed.

21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: A new labelling tool

Lipoproteins are important biological components. However, they have few convenient fluorescent labelling probes currently reported, and their physiological reliability can be questioned. We compared the association of two fluorescent cholesterol derivatives, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), to serum lipoproteins and to purified HDL and LDL. Both lipoproteins could be stably labelled by Pyr-met-Chol, but virtually not by NBD-Chol. At variance with NBD-Chol, LCAT did not esterify Pyr-met-Chol. The labelling characteristics of lipoproteins by Pyr-met-Chol were well distinguishable between HDL and LDL, regarding dializability, associated probe amount and labelling kinetics. We took benefit of the pyrene labelling to approach the structural organization of LDL peripheral hemi-membrane, since Pyr-met-Chol-labelled LDL, but not HDL, presented a fluorescence emission of pyrene excimers, indicating that the probe was present in an ordered lipid micro-environment. Since the peripheral membrane of LDL contains more sphingomyelin (SM) than HDL, this excimer formation was consistent with the existence of cholesterol- and SM-enriched lipid microdomains in LDL, as already suggested in model membranes of similar composition and reminiscent to the well-described “lipid rafts” in bilayer membranes. Finally, we showed that Pyr-met-Chol could stain cultured PC-3 cells via lipoprotein-mediated delivery, with a staining pattern well different to that observed with NBD-Chol non-specifically delivered to the cells.

The mechanically induced color change from UV to visible region

The branched amidoamine molecule 1 with different chromophores of a rhodamine B and two pyrenyl moieties at each side was synthesized. The luminescent properties of 1 with the color change from bright blue (UV region) to reddish (visible region) under mechanical stimulus were observed, which was attributed to the emission of pyrene excimer II, with a sandwich packing mode, and the chemical reaction of rhodamine B from a spirolactam to a ring opened amide.

A Continuous Fluorometric Assay for Trypsin Based on Melittin and the Noncovalent-binding-induced Pyrene Excimer

Abstract A continuous fluorometric assay system based on melittin and a pyrene derivative 1 for trypsin and its inhibitor screening is successfully developed by taking advantage of the noncovalent-binding-induced pyrene excimer. The 1–melittin assembly and its disassembly after further addition of trypsin are confirmed by the fluorescence changes at 475 nm. Such a system can be applied to other proteases of melittin, and essentially, the concept based on the noncovalent-binding-induced continuous fluorometric assay can be extended to other biochemical analyses.

Reaction-based Hg2+ signaling by excimer–monomer switching of a bis-pyrene dithioacetal

A novel reaction-based probe for fluorescence signaling of Hg 2+ ions was developed. Selective Hg 2+ -induced cleavage of a dithioacetal resulting in switching from pyrene excimer to monomer emission was used for the signaling. Changes in excimer and monomer emissions of pyrene were readily employed for ratiometric signaling of Hg 2+ ions in aqueous acetonitrile. Selective signaling of Hg 2+ ions over other common metal ions was observed with a detection limit of 9.8 × 10 −7 M.

Urea effect on aggregation and adsorption of sodium dioctylsulfosuccinate in water

Understanding the mechanism that controls the folding/unfolding of proteins in the presence of urea continues to be a subject of research, and since micelles mimic biological aggregates, equal importance has been given to the study of surfactants in the presence of urea. Despite several studies on the effect of urea on the behavior of reverse micelles and microemulsions based on sodium dioctylsulfosuccinate (AOT), the urea effect on AOT regular micelles has not been investigated and hence it is studied herein by using surface tension, steady-state fluorescence, and dynamic light scattering methods. The effect of urea on the behavior of AOT is found to be different below and above 1.0molkg−1 urea (cu). The critical micelle concentration (cmc) is almost independent of urea concentration below cu, whereas it increases with increasing urea amount above cu. In AOT+urea aqueous solution below cu, added NaCl at a particular critical concentration (c*) induces sudden increase in the values of (i) counterion binding constant, (ii) aggregation number, (iii) fluorescence intensity ratio of pyrene excimer to monomer, and (iv) hydrodynamic diameter of AOT aggregate, whereas such changes are suppressed by urea above cu. NaCl-induced shape change in AOT micelle takes place if urea concentration is below cu, but hindered above cu. The adsorption behavior of AOT at the air–solution interface as a function of NaCl is also found to be different below and above cu. The urea effect is explained in terms of increase in the polarity of the medium, better solvation of head groups and counterions, and weakening of head group–head group and head group–counterion interactions.

Organic-Inorganic Hybrid Mesoporous Materials as Regenerable Sensing Systems for the Recognition of Nitroaromatic Explosives.

Fluorescent organic-inorganic mesoporous hybrid materials have been prepared and characterised, and their behaviour against nitroaromatic explosives have been tested. MCM-41 silica was used as an inorganic scaffold and pyrene (P derivative containing trialkoxysilane moieties), dansyl and fluorescein (D and F derivatives also containing trialkoxysilane groups, respectively) fluorophores have been anchored on hybrid materials by a co-condensation method to obtain a homogenous distribution of dyes into the pores of the support. Six sensing materials have been prepared, of which SP, SD, SF were hydrophilic and SPh, SDh, SFh were hydrophobic. Template-free hydrophilic materials (SP, SD, SF) were obtained after repeated NH4 NO3 /ethanol extractions under temperature from as-synthesised (MP, MD and MF supports, respectively) solids. Hydrophobic materials (SPh, SDh, SFh) were prepared by using excess 1,1,1,3,3,3-hexamethyldisilazane with template-free hydrophilic (SP, SD and SF) materials. The six final materials displayed the typical emission bands of the grafted fluorophores. In particular, SP and SPh show the typical pyrene monomer (370-420 nm) and excimer (430-600 nm) emissions. SD and SDh exhibit the broad dansyl fluorescence band in the 450-600 nm range, whereas solids SF and SFh present sharp fluorescein emission centred at 525 nm. The fluorescent behaviour of the six final materials was tested in the presence of explosives (pentaerythritol tetranitrate (PETN), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), nitrobenzene (NB), 1,3,5-trinitrobenzene (TNB), 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), 2,4,6-trinitrophenylmethylnitramine (Tetryl) and picric acid (PA)). Only nitroaromatic compounds were able to induce emission quenching. As a general trend, the quenching degree depended on the nature of the final material. The best response was obtained with explosives PA and Tetryl, which were able to significantly quench the emission of the sensing supports. The observed quenching was ascribed to the π-π stacking interactions between the electron-donor fluorophores and the electron-withdrawing nitroaromatic explosives. When using SPh for Tetryl and PA, the limits of detection were 8.5 and 1.4 ppm, respectively, whereas they were 14.4 and 1.2 ppm for SDh. Principal component analysis algorithms were applied to the fluorescence measurements taken with the six hybrid materials and the seven explosives. The obtained score plot showed well-defined clusters for the seven explosives tested. Finally, solid SDh was applied to detect trace amounts of Tetryl in soil samples with good results.

Modulating the nanorods protrusion from poly(allylamine hydrochloride)-g-pyrene microcapsules by 1-pyrenesulfonic acid sodium salt

It was found previously that the Schiff base bonds in poly(allylamine hydrochloride)-g-pyrene (PAH-Py) microcapsules (MCs) are hydrolyzed at pH2 within 1h, leading to disassembly of the MCs and protrusion of pyrene aldehyde (Py) nanorods (NRs) on the capsule surface. Herein, we found a new way to modulate the protrusion of NRs by addition of 1-pyrenesulfonic acid sodium salt (PySO3Na). Along with the increase in PySO3Na to Py molar ratio in the MCs solution, the protrusion of NRs was progressively blocked and even inhibited at a ratio of 2.3, and at this condition, the microcapsules were stable under pH2 for 24h. After the composite microcapsules with excess PySO3Na were washed with a pH10 solution and then incubated in a pH2 solution, the NRs could be protruded from the MCs again. The fluorescence peak position of the PAH-Py/PySO3Na MCs gradually red-shifted with a decrease in pH value, and a sharp transition occurred at pH3.6, demonstrating the formation of pyrene excimers between the PySO3Na small molecules and the pendant Py groups on the PAH chain. The formed excimers take the role of blocking the self-assembly of cleaved Py molecules instead of inhibiting the hydrolysis of the Schiff base, whereas the MCs were stabilized by the charge interaction between PySO3Na and PAH backbone and the hydrophobic interaction between the pyrene rings.

Appropriate excitation wavelength removes obfuscations from pyrene excimer kinetics and mechanism studies

The experimental procedures of time-resolved laser photolysis studies of pyrene excimer formation in solution have been scrutinized and the appropriate modifications have been implemented. Contrary to the experimental methods applied in all related previous work, the selection of a suitable excitation wavelength (such that the corresponding pyrene absorbance is less than 0.5 absorbance units) utilized in our study results in simple homogeneous kinetics. Consequently, the rate parameters obtained and the mechanism proposed differ significantly from those published previously. The rate constant values of the unimolecular decay of the pyrene monomer, the unimolecular decay of the pyrene excimer, and the excimer formation in decane solution (η = 0.860 mPa s) at 25 °C are (2.38 ± 0.01) × 10(6) s(-1), (2.78 ± 0.02) × 10(7) s(-1), and (3.11 ± 0.06) × 10(9) M(-1) s(-1), respectively. The dissociation of the excimer to form a singlet excited state pyrene and a ground state pyrene was shown to be negligible. The energies of activation corresponding to the monomer and excimer unimolecular decays were found to be 2.51 ± 0.07 and 25.7 ± 0.7 kJ mol(-1), respectively. Also, our temperature resolved laser photolysis data revealed that the excimer formation has a negative energy of activation equal to -11.2 ± 0.5 kJ mol(-1). This unique phenomenon may be attributed to steric effects in the collision of the reactants. The current findings are important for the correct data analysis and interpretation in many applications of the pyrene excimer.

A quencher-free molecular beacon design based on pyrene excimer fluorescence using pyrene-labeled UNA (unlocked nucleic acid)

A quencher-free molecular beacon capable of generating pyrene excimer fluorescence has been constructed using strategically positioned pyrene-UNA monomers. Hybridization of a fully complementary RNA target was accompanied by a pyrene excimer emission increase of more than 900%, and detection of RNA in living cells was demonstrated.

A Butterfly-Shaped Pyrene Derivative of Cholesterol and Its Uses as a Fluorescent Probe

A butterfly-shaped pyrene derivative of cholesterol, namely, N,N'-(ethane-1,2-diyl)-bis(N-(2-(chol-amino)ethyl)pyrene-1-sulfonamide) (ECPS), has been designed and synthesized. Solvent effect studies revealed that in good solvents such as n-hexane, benzene, and 1,4-dioxane, the profile of the fluorescence emission of the compound is characterized by pyrene monomer emission, but in poor solvent such as water, the emission is dominated by pyrene excimer emission. Quantitatively speaking, the ratio of the excimer emission to monomer emission changes from 50 to 0 when ECPS is dissolved in water and n-hexane, respectively. In contrast, for a commonly used polarity probe pyrene, the ratio of I3/I1 varies only from ~0.6 to ~1.7, where I3 and I1 stand for the intensities of the fluorescence emission at peak 3 and peak 1, respectively. This value suggests that a more powerful discriminating ability of the new compound in polarity sensing. Furthermore, unlike the main components of the compound, pyrene and cholesterol, its main chain is composed of multiple hydrophilic structures, and it is this structure that makes the emission of the compound in organic solvents sensitive to the presence of water. Accordingly, the applicability of the compound in determination of the trace amount of water in some organic solvents was evaluated. As expected, the detection limit of the compound toward water in acetonitrile reaches 7 ppm, a result never reached before. Furthermore, the fluorescence emission of the compound is also sensitive to viscosity variation. Therefore, it is assumed that ECPS may be used both as a polarity probe and a viscosity probe. On the bases of a series of steady-state and time-resolved fluorescence, as well as dynamic light scattering studies, a structural model was proposed to rationalize the fluorescence behavior of the compound in different solvents and its polarity and viscosity probing performances.

External Electric Field Effect on Fluorescence Spectra of Pyrene in Solution

Electrophotoluminescence (E-PL) spectra, i.e., plots of the electric-field-induced change in photoluminescence intensity as a function of wavenumber, have been measured for pyrene solution. At high concentrations of pyrene where excimer fluorescence is observed along with the monomer fluorescence emitted from the locally excited state, both excimer fluorescence and monomer fluorescence are enhanced by application of electric fields. The results show that the nonradiative decay process at the excimer emitting state is decelerated by application of electric fields. It is also found that molecular polarizability of pyrene excimer is larger than that of pyrene monomer in the ground state by ~270 ± 90 Å(3), based on the analysis of the Stark shift of the excimer fluorescence.

A Locked Nucleic Acid-Based Nanocrawler: Designed and Reversible Movement Detected by Multicolor Fluorescence

Herein we introduce a novel fluorescent LNA/DNA machine, a nanocrawler, which reversibly moves along a directionally polar complementary road controlled by affinity-enhancing locked nucleic acid (LNA) monomers and additional regulatory strands. Polyaromatic hydrocarbon (PAH) dyes attached to 2'-amino-LNA monomers are incorporated at four stations of the system, enabling simple detection of the position of the nanocrawler via a step-specific color signal. The sensing is provided by highly sensitive, chemically stable, and photostable PAH LNA interstrand communication systems, including pyrene excimer formation and pyrene-perylene interstrand Förster resonance energy transfer. We furthermore demonstrate that the nanocrawler selectively and reversibly moves along the road, followed by a bright and consistent fluorescence response for up to 10 cycles without any loss of signal.

ATP Alone Triggers the Outward Facing Conformation of the Maltose ATP-binding Cassette Transporter

The maltose transporter MalFGK(2) is a study prototype for ABC importers. During catalysis, the MalFG membrane domain alternates between inward and outward facing conformations when the MalK dimer closes and hydrolyzes ATP. Because a rapid ATP hydrolysis depends on MalE and maltose, it has been proposed that closed liganded MalE facilitates the transition to the outward facing conformation. Here we find that, in contrast to the expected, ATP is sufficient for the closure of MalK and for the conversion of MalFG to the outward facing state. The outward facing transporter binds MalE with nanomolar affinity, yet neither MalE nor maltose is necessary or facilitates the transition. Thus, the rapid hydrolysis of ATP observed in the presence of MalE and maltose is not because closed liganded MalE accelerates the formation of the outward facing conformation. These findings have fundamental implications for the description of the transport reaction.

Probing the Hydrophobic Interactions of a Series of Pyrene End-Labeled Poly(ethylene oxide)s in Aqueous Solution Using Time-Resolved Fluorescence

The hydrophobic association of a series of poly(ethylene oxide)s covalently labeled at both ends with pyrene (PEO(X)-Py2 where X represents the number average molecular weight (Mn) of the PEO chains equal to 2, 5, 10, and 16.5 kDa) in aqueous solutions was investigated at different polymer concentrations (CP) using steady-state and time-resolved fluorescence measurements. Phase separation was observed with PEO(2 kDa)-Py2 and PEO(5 kDa)-Py2 samples at high CP. The steady-state fluorescence spectra showed that the ratios of excimer-to-monomer fluorescence intensities (IE/IM) of all PEO samples remained constant when CP was below 4 × 10(-5) M and decreased dramatically with increasing PEO chain length due to a decrease in intramolecular pyrene excimer formation. The IE/IM ratio in this regime was found to scale as Mn(-2.3±0.2). For CP > 4 × 10(-5) M, pyrene excimer is formed by both intra- and intermolecular interactions and the IE/IM ratio increases linearly with increasing CP except for PEO(2 kDa)-Py2 which undergoes phase separation. The decays obtained at various polymer concentrations were fitted according to a "sequential model" (SM) which assumes that the pyrene excimer is formed in a sequential manner. The molar fractions of all excited pyrene species and the rate constants for pyrene excimer formation were determined from the global analysis of the monomer and excimer fluorescence decays. The fraction of pyrenes that formed excimer from ground-state pyrene aggregates (fE0) was found to increase with CP in the regime where the pyrene excimer is formed both intra- and intermolecularly and decrease with Mn in the regime where the pyrene excimer is formed only intramolecularly. The fraction of pyrene pendants subject to hydrophobic interactions were used to determine the hydrophobic capture radius (Rc) of pyrene in water from the distribution of PEO end-to-end distances. Rc was found to equal 2.2 ± 0.2 nm using fE0.

Metal ion-prompted pyrene–excimer formation via an anion-mediated process and its application for a ratiometric Zn2+ chemosensor with high selectivity over Cd2+

The combination of [(2,2′-dipicolylamino)butyl]pyrene (1) and pyrophosphate (PPi) was shown to be functional as a ratiometric Zn2+ ion fluorescent probe. This was changed to Zn2+-1 upon the addition of Zn2+, which was dimerized by a PPi-mediated self-assembly, leading to a relatively strong emission fluorescence with a large bathchromic shift. This chemosensor can detect Zn2+ in an aqueous solution of physiological pH with high selectivity toward Zn2+ over a variety of metal ions, including Cd2+, and has a low detection limit of 0.64μM in an aqueous buffer system.