Pyrene Excimer Research Articles

Pyrene Excimer for DNA Sensors

NSFC [20805038, 20620130427]; MOE [200803841013]; MOST [2007CB935603, 2007DFC40440, 2010CB732402]; US NIH

New pyrenyl fluorescent amphiphiles: synthesis and aggregation properties

Four cationic amphiphilic fluorescent probes were synthesized and fully characterized. All amphiphiles feature a pyrrolidinium headgroup and a hydrocarbon tail tagged with a pyrene residue, and differ in the connection between the headgroup and the tagged tail, and in the hydrophilic/hydrophobic balance, two being single tail surfactants and the others twin type surfactants. The aggregation features were investigated by electrical conductivity, fluorescence, dynamic light scattering and transmission electron microscopy experiments. The obtained results show that changes in the molecular structure of the monomer, by affecting the aggregation behavior, have a strong influence on the conformation of the fluorophore inside the aggregate, and hence on its fluorescence features in aggregating conditions. In particular, the formation of pyrene dimers and/or excimers was observed only for the twin type surfactants, thus suggesting that a second alkyl chain is crucial for warranting a specific orientation of the pyrene moiety suitable for dimers or excimers formation. Moreover, the different linkage of the pyrene moiety to the alkyl chain in the twin type surfactants results in a remarkable difference in the organization of the aggregates they form, in fact only in the aggregates formed by one of the two surfactants the pyrene moiety is exposed at the lipid–water interface.

Signal control by self-assembly of fluorophores in a molecular beacon—a model study

Pyrene excimer fluorescence is efficiently regulated through formation of π-stacked aggregates between dialkynylpyrene (Y) and perylenediimide (E) residues located in the stem region of a molecular beacon (MB). The building blocks form organized, multichromophoric complexes in the native form. Hybridization to the target results in a conformational reorganization of the chromophores. The nature of the aggregates was investigated by changing the number of chromophores and natural base pairs in the beacon stem. The formation of different types of complexes (EYEY→YEY→EY) is revealed by characteristic spectroscopic changes. The data show that signal control is an intrinsic property of the interacting chromophores. The directed assembly of non-nucleosidic chromophores can be used for the generation of an on/off switch of a fluorescence signal. The concept may find applications in various types of light-based input/output systems.

A highly selective ratiometric fluorescent chemosensor for Ag+ based on a rhodanineacetic acid–pyrene derivative

A highly selective ratiometric fluorescent Ag+ chemosensor RAAP which featured Ag+-induced enhancement of fluorescence emission and blue-shift of the UV-vis spectrum (64 nm) was developed. Both of the changes are due to the Ag+-induced conformational changes from a pyrene monomer to a pyrene excimer and the formation of a crossing configuration. The fluorescence detection limit of Ag+ can reach 1.0 × 10−7 M, which complies with the standards of US EPA and World Health Organization (WHO) for drinking water (5.0 × 10−7 M). RAAP is organic solvent-stable and pH-stable and its spectral response toward Ag+ is found to be reversible.

Pyrene‐Excimer Probes Based on the Hybridization Chain Reaction for the Detection of Nucleic Acids in Complex Biological Fluids

Make it a double: Complementary probes with two pyrene labels were engineered for the amplification of a target DNA sequence. In the stem-closed conformation of the probes in the absence of the target, the two pyrene moieties were separated. The target propagated hybridization chain reactions to bring pyrene moieties on neighboring probes close together to form fluorescent pyrene excimers (see picture).

An Aptamer-based and Pyrene-labeled Fluorescent Biosensor for Homogeneous Detection of Potassium Ions

A simple and homogeneous method based on aptamer and pyrene moieties for the detection of K(+) was developed. The aptamer was labeled by pyrene moiety at 3' end as the molecular recognition element. In the presence of K(+), the complementary oligonucleotide labeled by pyrene moiety at 5' end was displaced from the aptamer, which was accompanied by a dramatic decrease of the excimer fluorescence of pyrene. However, the excimer fluorescence remains in the absence of the target. With optimum conditions, relative changes of the pyrene excimer fluorescence intensity were proportional to the concentrations of K(+) in the range of 6.3 × 10(-4) to 1.0 × 10(-2) M with a detection limit of 5.0 × 10(-4) M. Importantly, in the presence of Na(+), NH(4)(+), Mg(2+) and Ca(2+) cations of biological fluids, this method was able to detect K(+) with high selectivity. In a word, the assay seems to have great potential applications, especially in biological fluids due to its simplicity, specificity and homogeneous detection.

Competition-Mediated Pyrene-Switching Aptasensor: Probing Lysozyme in Human Serum with a Monomer-Excimer Fluorescence Switch

Lysozyme (Lys) plays crucial roles in the innate immune system, and the detection of Lys in urine and serum has considerable clinical importance. Traditionally, the presence of Lys has been detected by immunoassays; however, these assays are limited by the availability of commercial antibodies and tedious protein modification and prior sample purification. To address these limitations, we report here the design, synthesis, and application of a competition-mediated pyrene-switching aptasensor for selective detection of Lys in buffer and human serum. The detection strategy is based on the attachment of pyrene molecules to both ends of a hairpin DNA strand, which becomes the partially complementary competitor to an anti-Lys aptamer. In the presence of target Lys, the aptamer hybridizes with part of the competitor, which opens the hairpin such that both pyrene molecules are spatially separated. In the presence of target Lys, however, the competitor is displaced from the aptamer by the target, subsequently forming an initial hairpin structure. This brings the two pyrene moieties into close proximity to generate an excimer, which, in turn, results in a shift of fluorescence emission from ca. 400 nm (pyrene monomer) to 495 nm (pyrene excimer). The proposed method for Lys detection showed sensitivity as low as 200 pM and high selectivity in buffer. When measured by a steady-state fluorescence spectrum, the detection of Lys in human serum showed a strong fluorescent background, which obscured detection of the excimer signal. However, time-resolved emission measurement (TREM) supported the potential of the method in complex environments with background fluorescence by demonstrating the temporal separation of probe fluorescence emission decay from the intense background signal. We have also demonstrated that the same strategy can be applied to the detection of small biomolecules such as adenosine triphosphate (ATP), showing the generality of our approach. Therefore, the competition-mediated pyrene-switching aptasensor is promising to have potential for clinical and forensic applications.

Pyrene Excimer-Based Calix[4]arene FRET Chemosensor for Mercury(II)

A novel calix[4]arene derivative locked in the 1,3-alternate conformation (2) bearing two pyrene and rhodamine fluorophores was synthesized as a selective sensor for the Hg(2+) ion. The sensoring is based on FRET from pyrene excimer emissions to ring-opened rhodamine absorption upon complexation of the Hg(2+) ion. Addition of Hg(2+) to a mixed solution of 2 gave significantly enhanced fluorescence at ∼576 nm via FRET with excitation at 343 nm. We also found that the pyrene excimer emissions formed by the intramolecular π-π interactions are more effective in obtaining strong FRET bands than those by intermolecular π-π interactions.

DNA Staining in Agarose Gels with ZN2+-Cyclen-Pyrene

A pyrene-labeled Zn2+-cyclen complex for the staining of DNA in agarose gels is reported. The metal chelate coordinates reversibly to the DNA phosphate backbone, which induces the formation of pyrene excimers. The typical pyrene excimer emission is used for the detection of the DNA. Staining is limited to agarose gels and is less sensitive than ethidium bromide, but DNA amounts as low as 10 ng and short DNA strands (∼300 b.p.) are detectable. Gel extraction as a standard technique in molecular biology was successfully performed after staining with Zn2+-cyclen-pyrene. Cytotoxicity tests on HeLa and V-79 cells reveal that the zinc-cyclen pyrene probe is significant less toxic compared to ethidium bromide.

Kinetic analysis of the interaction between poly(amidoamine) dendrimers and model lipid membranes

We used fluorescence spectroscopy and surface tensiometry to study the interaction between low-generation (G1 and G4) poly(amidoamine) (PAMAM) dendrimers, potential vehicles for intracellular drug delivery, and model lipid bilayers. Membrane association of fluorescently labeled dendrimers, measured by fluorescence anisotropy, increased with increasing size of the dendrimer and with increasing negative charge density in the membrane, indicating the electrostatic nature of the interaction. When the membrane was doped with pyrene-labeled phosphatidyl glycerol (pyrene-PG), pyrene excimer fluorescence demonstrated a dendrimer-induced selective aggregation of negatively charged lipids when the membrane was in the liquid crystalline state. A nonlinear Stern–Volmer quenching of dendrimer fluorescence with cobalt bromide suggested a dendrimer-induced aggregation of lipid vesicles, which increased with the dendrimer's generation number. Surface tensiometry measurements showed that dendrimers penetrated into the lipid monolayer only at subphysiologic surface pressures (<30mN/m). We conclude that the low-generation PAMAM dendrimers associate with lipid membranes predominantly electrostatically, without significantly compromising the bilayer integrity. They bind stronger to membranes with higher fluidity and lower surface pressure, which are characteristic of rapidly dividing cells.

Ultrafast Dynamics of 1-Ethynylpyrene-Modified RNA: A Photophysical Probe of Intercalation

The photophysics of pyrene attached to an adenine base within RNA single strands and duplexes is examined with respect to the position of the pyrene within the strand and the number of pyrenes attached to one duplex. Compounds with pyrenes intercalating sequence specifically are examined, as well as a doubly modified compound, where the two pyrenes are located close enough to each other for significant excimer interaction. Femtosecond transient absorption measurements and time correlated single photon counting measurements allow a thorough examination of the local influences on the pyrene photophysics. Our results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties and lifetimes that are coupled only via the excited state as a gateway. One of them is a neutral pyrene-adenine excited state, S*, while the second one is connected to an excited charge transfer state, S*(CT). In all compounds, an ultrafast sub-ps decay from a higher excited state into the lowest excited state S* occurs, and an excited charge transfer species S*(CT) is formed within picoseconds. The fluorescence behavior of the pyrene-modified adenine, however, is strongly dependent on RNA conformation. Both S* and S*(CT) states are fluorescent, and decay within hundreds of picoseconds and approximately 2 ns, respectively. The ratio between S* and S*(CT) fluorescence depends strongly on pyrene intercalation, and it is found that the S* state is quenched selectively upon intercalation of the pyrene into RNA. The doubly modified duplex exhibits an additional fluorescent state with a lifetime of 18.7 ns, which is associated with the pyrene excimer state. This state coexists with a significant population of the pyrene monomer, since the characteristic features of the latter can still be observed. Formation of the excimer occurs on femtosecond time scales. The pyrene label thus provides a sensitive tool to monitor the local structural dynamics of RNA with the chromophore acting as a molecular beacon.

Fluorescence Study of Intermolecular Interactions in Diluted Aqueous Solutions of Some Cationic Amphiphilic Polysaccharides

The intermolecular hydrophobic association in diluted aqueous solutions of some cationic amphiphilic polysaccharides was investigated using fluorescence techniques. Dextran and dextran carrying N-alkyl-N,N-dimethyl-N(2-hydroxypropylene) ammonium chloride groups as side chains were single labeled with pyrene or naphthalene. The intensity of the pyrene excimer peak and the ratio I3/I1 determined from the fluorescence emission spectra of pyrene-labeled amphiphilic polymer increased with increasing polymer concentration and were higher than in the solution containing pyrene-labeled dextran. Emission spectra of diluted solution (0.008–0.2 g/dl) containing mixtures of pyrene and naphthalene single-labeled amphiphilic polymers proved the occurrence of a nonradiative energy transfer between labels at very a low polymer concentration (<10−2 g/dl). The energy transfer was not observed in mixtures of single-labeled unmodified dextran. All these results suggest that the intermolecular hydrophobic association of alkyl substituents takes place at a very low concentration of amphiphilic polymers.

Fluorescent and Electrochemical Sensing of Polyphosphate Nucleotides by Ferrocene Functionalised with Two ZnII(TACN)(pyrene) Complexes

The [Fc-bis{Zn(II)(TACN)(Py)}] complex, comprising two Zn(II)(TACN) ligands (Fc=ferrocene; Py=pyrene; TACN=1,4,7-triazacyclononane) bearing fluorescent pyrene chromophores linked by an electrochemically active ferrocene molecule has been synthesised in high yield through a multistep procedure. In the absence of the polyphosphate guest molecules, very weak excimer emission was observed, indicating that the two pyrene-bearing Zn(II)(TACN) units are arranged in a trans-like configuration with respect to the ferrocene bridging unit. Binding of a variety of polyphosphate anionic guests (PPi and nucleotides di- and triphosphate) promotes the interaction between pyrene units and results in an enhancement in excimer emission. Investigations of phosphate binding by (31)P NMR spectroscopy, fluorescence and electrochemical techniques confirmed a 1:1 stoichiometry for the binding of PPi and nucleotide polyphosphate anions to the bis(Zn(II)(TACN)) moiety of [Fc-bis{Zn(II)(TACN)(Py)}] and indicated that binding induces a trans to cis configuration rearrangement of the bis(Zn(II)(TACN)) complexes that is responsible for the enhancement of the pyrene excimer emission. Pyrophosphate was concluded to have the strongest affinity to [Fc-bis{Zn(II)(TACN)(Py)}] among the anions tested based on a six-fold fluorescence enhancement and 0.1 V negative shift in the potential of the ferrocene/ferrocenium couple. The binding constant for a variety of polyphosphate anions was determined from the change in the intensity of pyrene excimer emission with polyphosphate concentration, measured at 475 nm in CH(3)CN/Tris-HCl (1:9) buffer solution (10.0 mM, pH 7.4). These measurements confirmed that pyrophosphate binds more strongly (K(b)=(4.45+/-0.41) x 10(6) M(-1)) than the other nucleotide di- and triphosphates (K(b)=1-50 x 10(5) M(-1)) tested.

A Study of the Dynamics of the Branch Ends of a Series of Pyrene-Labeled Dendrimers Based on Pyrene Excimer Formation

A series of pyrene-labeled dendrimers were prepared from generation n = 1 to n = 4 where the pyrenes were attached to the end groups of the dendrimers. Pyrene excimer formation was monitored by steady-state and time-resolved fluorescence spectroscopy as a function of generation number and in terms of the I(E)/I(M) ratio and the average rate constant of excimer formation <k>. To account for the unconventional distribution of pyrene labels which were neither randomly distributed throughout the macromolecule nor limited to just two units which are the only two pyrene-labeling schemes that can be dealt with in a straightforward manner, a Model Free (MF) analysis was applied to the global analysis of the fluorescence decays. Within experimental error, the I(E)/I(M) ratios and <k> obtained from, respectively, the steady-state fluorescence spectra and the time-resolved fluorescence decays were found to increase linearly with increasing generation number. This result is inconsistent with the fact that both the I(E)/I(M) ratio and <k> are proportional to the local concentration of pyrene inside the dendrimer ([Py](loc)) which is not expected to increase with generation number if the excited pyrene is assumed to diffuse freely throughout the dendrimer interior. Since the core-dense model predicts that the dendrimer terminal ends can occupy any position throughout the dendrimer interior, these results suggest that excimer formation between the pyrene-labeled ends is enhanced due to the branched nature of the dendrimer.

Hg2+-Selective Ratiometric and “Off−On” Chemosensor Based on the Azadiene−Pyrene Derivative

A new pyrene-based derivative bearing an azadiene group was synthesized as a ratiometric chemosensor for Hg(2+) in aqueous acetonitrile solution. The "off-on" type signaling behavior of the fluoroionophore is due to the metal ion induced conformational changes from the weak pyrene monomer emissions to strong pyrene excimer emission.

Steady state and time-resolved studies of pyrene in solution and in single microcrystals

Single microcrystals of pyrene have been studied by steady state and time resolved fluorescence microscopy. The fluorescence spectra of microcrystals exhibit vibrational structure unlike the broad spectrum observed in pyrene excimer. A risetime is observed in the decay curves of the concentrated solutions, indicating the excimer formation. In contrast, the fluorescence decay profiles of the single microcrystals are nonexponential in nature and the decay times vary with their size and the wavelength of emission. This behaviour has been explained mainly by considering the pyrene dimer stabilized in the ground state.

Enclosure of Secondary Chirality Based on Highly‐Oriented Lipid Aggregates into a Polymer Sheet by Photo‐Induced Polymerization of Polymerizable Monomer Gels

AbstractThe L‐glutamide derivative with double long‐chain alkyl groups and a pyrenyl head group (Pyr‐lipid) worked as organogelator in benzene and cyclohexane. Gelation was brought about through network formation with well‐developed fibrous aggregates based on lipid membrane‐like highly‐oriented structures. Gelation was observed in polymerizable monomers such as methyl methacrylate (MMA) and styrene (St). Extremely intense CD spectra were observed at the absorption band of pyrenyl group in a monomer solution at gel‐forming temperature, but the intensity decreased remarkably at the sol‐forming temperature. The transparent solid sheet was obtained by photo‐irradiation to the MMA gel in a quartz cell. More than 99% of MMA was polymerized even in the gel state and the weight‐average molecular weight (Mw) of the resultant polymer was 7.2 × 104 g mol−1. CD spectra indicated that chirally‐oriented structures in the gel state were maintained after polymerization of MMA and were stable at the temperature above the original gel‐to‐sol transition temperature. Fluorescence spectra of Pyr‐lipid in the monomer solutions supported that the pyrene excimer was formed in the lipid aggregates and the stabilization of the excimer formation was achieved by polymerization of MMA in the gel state.

Alterations in membrane fluidity and dynamics in experimental colon cancer and its chemoprevention by diclofenac

We examined the role of membrane fluidity and dynamics as important early events in the carcinogenic transformation of colonic epithelial cells. 1,2-Dimethylhydrazine dihydrochloride (DMH) was used to induce initial stages of colon cancer and diclofenac was used for chemoprevention. To determine alterations of membrane fluidity of rat colonic epithelial cells, fluidity (inverse of fluorescence polarization) and order parameter were studied with 1,6-diphenylhexatriene (DPH) polarization. Order parameter as well as fluorescence polarization was found to be significantly decreased, thus demonstrating an increase in the fluidity of the membrane. To further confirm the fluidity changes, microviscosity of the cell membrane was studied using pyrene excimer formation, which showed a significant decrease in microviscosity and hence elevated membrane fluidity (translational diffusion). The colonocytes were stained with merocyanine 540 (MC540) to further elaborate the changes in membrane fluidity and lipid packing. The increased number of colonocytes showing high MC540 fluorescence pointed towards the wide spaces and hence, high fluidity in the membrane after DMH treatment. Membrane dynamics studies, i.e., lipid phase separation and membrane phase state were carried out using N-NBD-PE and Laurdan, respectively. We saw a transition from the gel to a more liquid crystalline state of the membrane in the Laurdan experiment. Further more percentage quenching (%Q) value of N-NBD-PE showed less phase separation (or domain formation). Diclofenac co-administration with DMH was successful in reverting the changes observed, confirming the role of these anti-inflammatory drugs in considerable lipid affinity and consequently in the chemoprevention of early stages of colon cancer.

How switching the substituent of a pyrene derivative from a methyl to a butyl affects the fluorescence response of polystyrene randomly labeled with pyrene

A series of polystyrenes randomly labeled with 1-pyrenebutanol were prepared by copolymerizing styrene and 1-pyrenebutylacrylate yielding the CoBuE–PS series. Solutions of CoBuE–PS were prepared in nine organic solvents having viscosities ranging from 0.36 to 5.5 mPa·s and the fluorescence spectra and pyrene monomer and excimer fluorescence decays were acquired. Analysis of the fluorescence spectra yielded the IE/IM ratio, whereas analysis of the fluorescence decays with the fluorescence blob model (FBM) yielded the parameters N blobo , &lt;kblob × Nblob&gt; , and k blobo . These parameters were compared to those obtained with two other series of pyrene-labeled polystyrenes, which had been studied earlier, namely CoA–PS and CoE–PS where pyrene was attached to the polymer backbone via a methylamide and benzyl methylether linker, respectively. Although the parameters IE/IM, N blobo , &lt;kblob × Nblob&gt;, and k blobo took different values according to the specific nature of the linker connecting pyrene to the polystyrene backbone, they exhibited trends that were quite similar for all the pyrene-labeled polystyrene constructs. The excellent agreement between the parameters retrieved for the three different types of pyrene-labeled polystyrenes suggests that the FBM accounts satisfyingly for differences in the nature of the label used, while still retrieving information pertinent to the polymer of interest.

Thymidine Nucleotide Sensor

Simple methods of sensing nucleo­tides in aqueous environments may provide useful insights into biological pathways and could lead to novel methods in diagnosis of various diseases. The authors report a sensory molecule composed of a zinc(II)-cyclen core with pendant pyrene groups. When thymidine nucleotide binds this molecule, the two pyrene groups are brought together, leading to a turn-on of pyrene excimer emission (470 nm) with concomitant turn-off of pyrene monomer emission (375 nm).