Time-resolved Fluorescence DNA-based Sensors for Reducing Background Fluorescence of Environment.

The fluorescence assay is one of the popular methods that is applied for detection of different targets. However, this method may show low sensitivity and high background in biological samples due to the natural fluorescence of different compounds in complicated samples. In addition, it inevitably affects the detection results accuracy. A fundamental solution to this problem is the use of the time-resolved fluorescence technique (TRF). The main component of this technique is the use of long fluorescence lifetime reagents. In this review, various time-resolved fluorescent reagents such as complexes of lanthanide ions, lanthanide-doped inorganic nanoparticles; Mn-doped ZnS quantum dots (QDs) and pyrene excimer are introduced. Moreover, TRF sensors, especially TRF aptasensors (DNA-based sensors) are discussed. This review will give new ideas for researchers to develop novel high-sensitive TRF sensors that can remove or decrease background fluorescence and use them for the detection of various targets in complicated samples without treatment.

Rapid sensitive fluorescence detection of cadmium (II) with pyrene excimer switching aptasensor.

Heavy metal cadmium (II) contamination often occurs, causing great health risk to human due to high toxicity of cadmium (II). Rapid, sensitive and simple detection of cadmium (II) are of great importance in environmental monitoring. Taking advantage of aptamer in specific recognition, easy modification, and capability of binding-induced structure change, here we reported a simple fluorescent sensor with rapid and sensitive response for Cd2+ using aptamer pyrene excimer switch. The aptamer was labeled with dual pyrene molecules at two ends of the sequence. The binding of Cd2+ to this aptamer probe brought the pyrene labels into close proximity and enhanced formation of a pyrene excimer, which generated increased fluorescence at 485 nm. By measuring the fluorescence of pyrene excimer, we achieved detection of Cd2+ with this aptasensor. Under the optimum experimental conditions, the detection limit of Cd2+ reached nanomolar levels. This method was selective and allowed for the detection of Cd2+ in tap water. This fluorescence aptasensor is promising for rapid detection of Cd2+ in broad applications.

Cyclodextrins with Multiple Pyrenyl Groups: An Approach to Organic Molecules Exhibiting Bright Excimer Circularly Polarized Luminescence.

We report a simple and effective approach to organic molecules exhibiting bright circularly polarized luminescence (CPL) by combining a chiral cyclic molecular scaffold and multiple excimer-enabling moieties. An α-cyclodextrin (CyD) scaffold was modified with six pyrenyl groups to obtain pyrene-cyclodextrins (PCDs) in a one-step synthesis from commercially available compounds. The PCDs exhibited high molar extinction coefficients (ϵ≈105  M-1  cm-1 ), polarized emission with a good dissymmetry factor (|glum |≈10-2 ), and quantum yield (Φf ≈0.5). Owing to the excellent photophysical properties of the PCDs, the circularly polarized luminescence brightness (BCPL ) reached 340 M-1  cm-1 . Photophysical and chiroptical studies of the PCDs with only five pyrene units and with linkers of various lengths connecting the CyD with the pyrene units revealed that the formation of a pyrene excimer in a spatially crowded environment is crucial for CPL anisotropy. This study paves the way for the development of bright CPL organic molecules.

Host-Guest Complexation-Induced Aggregation Based on Pyrene-Modified Cyclodextrins for Improved Electronic Circular Dichroism and Circularly Polarized Luminescence.

Several γ-cyclodextrin (CD) derivatives mono- or di-substituted by pyrenes at the primary rim of the CD were demonstrated to aggregate into nano-strips in aqueous solutions, with the pyrene moieties interpenetrating into γ-CD cavities. The hydrophobic complexation-induced aggregation provides a rigid chiral environment for the pyrenes and leads to significant electronic circular dichroism (ECD) and circularly polarized luminescence (CPL) activities, giving unprecedently high gabs and glum values up to 4.3×10-2 and 5.3×10-2 , respectively. The aggregates lead to excimer emission with high quantum yields and show BCPL and BiCPL up to 338. 6 M-1 cm-1 and 169.3 M-1 cm-1 , respectively.

Articulated rods - a novel class of molecular rods based on oligospiroketals (OSK).

We developed a new type of molecular rods consisting of two (or more) rigid units linked by a flexible joint. Consequently we called these constructs articulated rods (ARs). The syntheses of ARs were carried out by a flexible and modular approach providing access to a number of compounds with various functionalizations in terminal positions. First applications were presented with pyrene, cinnamoyl and anthracenyl labelled ARs.

2'-Bispyrene-modified 2'-O-methyl RNA probes as useful tools for the detection of RNA: synthesis, fluorescent properties, and duplex stability.

The synthesis and properties two series of new 2'-O-methyl RNA probes, each containing a single insertion of a 2'-bispyrenylmethylphosphorodiamidate derivative of a nucleotide (U, C, A, and G), are described. As demonstrated by UV melting studies, the probes form stable complexes with model RNAs and DNAs. Significant increases (up to 21-fold) in pyrene excimer fluorescence intensity were observed upon binding of most of the probes with complementary RNAs, but not with DNAs. The fluorescence spectra are independent of the nature of the modified nucleotides. The nucleotides on the 5'-side of the modified nucleotide have no effect on the fluorescence spectra, whereas the natures of the two nucleotides on the 3'-side are important: CC, CG, and UC dinucleotide units on the 3'-side of the modified nucleotide provide the maximum increases in excimer fluorescence intensity. This study suggests that these 2'-bispyrene-labeled 2'-O-methyl RNA probes might be useful tools for detection of RNAs.

Pyrene-modified PNAs: Stacking interactions and selective excimer emission in PNA2DNA triplexes.

Pyrene derivatives can be incorporated into nucleic acid analogs in order to obtain switchable probes or supramolecular architectures. In this paper, peptide nucleic acids (PNAs) containing 1 to 3 1-pyreneacetic acid units (PNA1-6) with a sequence with prevalence of pyrimidine bases, complementary to cystic fibrosis W1282X point mutation were synthesized. These compounds showed sequence-selective switch-on of pyrene excimer emission in the presence of target DNA, due to PNA2DNA triplex formation, with stability depending on the number and positioning of the pyrene units along the chain. An increase in triplex stability and a very high mismatch-selectivity, derived from combined stacking and base-pairing interactions, were found for PNA2, bearing two distant pyrene units.

Glycogen ß-particles surface characterized by a combination of size exclusion chromatography and pyrene excimer fluorescence before and after ß-amylolysis.

Size exclusion chromatography (SEC) and pyrene excimer formation (PEF) experiments were conducted to characterize the local density profile inside a glycogen sample before (Glycogen) and after (Gly-ß-LD) treatment with ß-amylase. These experiments were conducted to assess whether the density at the periphery of the glycogen particles was very high to limit access to proteins involved in the metabolism of glycogen as predicted by the Tier model or low as suggested by the Gilbert model. SEC analysis indicated that the density inside the Glycogen and Gly-ß-LD samples remained constant with particle size and was not affected by ß-amylolysis. Analysis of the PEF experiments conducted on the Glycogen and Gly-ß-LD samples labeled with 1-pyrenebutyric acid showed that the particles have a dense interior and loose corona. The conclusions reached by the SEC and PEF experiments agree with the Gilbert model and have implications for the association of glycogen ß-particles into larger α-particles.

Metal Ion Responsive Luminescent Bio-Templated Co-Assemblies: Label-Free Detection of Multi-Metal Ions in Aqueous Media.

Recently, anionic bio-templates have emerged as promising platforms for designing dynamic and stimuli-responsive chromophoric assemblies capable of light harvesting in aqueous media thereby mimicking natural photosynthesis. Here, we present multi-metal ion-responsive luminescent co-assemblies between cationic pyrene-imidazolium amphiphile and anionic bio-templates (ATP, heparin, and DNA) in aqueous media. The anionic bio-templates enhance Förster resonance energy transfer (FRET) in the co-assemblies, with pyrene serving as an excellent donor for generating tunable multi-luminescent materials with embedded acceptor dyes. However, a significant loss in energy transfer towards acceptor dyes was observed in the presence of various metal ions, attributed to excimeric emission quenching facilitated by electron transfer between the pyrene chromophore and metal ions. Interestingly, detailed studies revealed that only ATP-based co-assemblies exhibited quenching phenomena in the presence of metal ions, contrasting with heparin and ctDNA co-assemblies. Additionally, label-free detection of multi-metal ions in aqueous environments, such as Fe2+, Fe3+, and Cu2+ ions, was successfully achieved with lower detection limits of 0.01 µM (3 ppb), 0.12 µM (30 ppb), and 0.58 µM (150 ppb) respectively. These co-assemblies hold significant promise for practical applications in environmental and biomedical sensing, enabling sensitive monitoring of metal ion concentrations.

Assembly of pyrene-modified DNA/RNA duplexes incorporating a G-rich single strand region.

The structural properties of a DNA/RNA duplex having a pyrene residue at the 5' end of DNA and a G-rich single strand region at the 3' end of RNA were studied in detail. Fluorescence and ultracentrifugation analyses indicated the formation of a complex containing four DNA/RNA duplexes, which required a pyrene residue, G-rich sequence, RNA-type backbone, and high salt concentration.

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.

Interior of glycogen probed by pyrene excimer fluorescence.

A glycogen sample from oyster (O) and another from corn (C) were fluorescently labeled with 1-pyrenebutyric acid to yield two series of pyrene-labeled glycogen samples (Py-Glycogen(O/C)). Analysis of the time-resolved fluorescence (TRF) measurements of the Py-Glycogen(O/C) dispersions in dimethyl sulfoxide yielded the maximum number () of anhydroglucose units (AGUs), that could separate two pyrene-labeled AGUs and still allow efficient pyrene excimer formation (PEF) between an excited and a ground-state pyrene. Molecular mechanics optimizations (MMOs) were conducted on a lattice of hexagonally close packed oligosaccharide helices to determine how the theoretical Nblobtheo varied as a function of the lattice density. Comparing and , obtained after integrating Nblobtheo along the local density profile ρ(r) across the glycogen particles, led to the conclusion that ρ(r) took a maximum value at the center of the glycogen particles contrary to expectations based on the Tier Model.

Persistence Length of PEGMA Bottle Brushes Determined by Pyrene Excimer Fluorescence.

Seven pyrene-labeled poly(oligo(ethylene glycol) methyl ether methacrylate)s (PyEG5-PEGnMAs) were prepared with n = 0, 3, 4, 5, 7, 9, and 19 ethylene glycol units by copolymerizing a small amount of penta(ethylene glycol) 1-pyrenemethyl ether methacrylate with an EGnMA monomer. The conformation of the PyEG5-PEGnMA polymers evolved from a random coil for PyEG5-PEG0MA or poly(methyl methacrylate) to a polymeric bottle brush (PBB) architecture with increasing side chain length. The fluorescence decays of the PyEG5-PEGnMA samples were fitted according to the fluorescence blob model (FBM) whose parameters were used, in combination with the Kratky-Porod equation, to calculate the persistence length of these polymers. The persistence lengths obtained from the PEF experiments were found to increase with the square of the number (NS) of non-hydrogen atoms in the side chain as expected theoretically. The persistence lengths found with the PyEG5-PEGnMA samples in DMF also matched those found earlier for another series of PEGnMA samples labeled with 1-pyrenebutanol. The good agreement found between the persistence lengths obtained with the PEGnMA samples labeled with two different pyrene derivatives illustrates the robustness of the method and its applicability for measuring the unknown persistence length of polydisperse polymer samples.

ATP-Enhanced Multistimuli-Responsive Förster Resonance Energy Transfer in Organic Coassemblies.

In recent years, ATP has emerged as an anionic biocomponent for the design of dynamic and stimuli-responsive supramolecular assemblies. Herein, we present ATP-enhanced Förster resonance energy transfer (FRET) in the coassemblies of pyrene-imidazolium amphiphiles with pyrene acting as an excellent donor for the coembedded acceptor dyes to generate tunable multiluminescent materials in aqueous solutions and in polymer and solid films. We achieved high energy transfer efficiency up to 95% even at a donor/acceptor (D/A) ratio of 100:1. By a simple variation of the D/A ratio, emission covering almost the whole range of the visible spectrum from blue to red including white light was obtained in solution and in solid and polymer films. Furthermore, the systems exhibited FRET ON/OFF features controlled by various stimuli such as temperature, pH, and metal ions. Most notably, a ratiometric and linear luminescence response to temperature and pH was observed. The stimuli-responsive tunable solid-state emission was further exploited in encryption-decryption applications.

Cluster Size of Amylopectin and Nanosized Amylopectin Fragments Characterized by Pyrene Excimer Formation.

Amylopectin from waxy corn and the three nanosized amylopectin fragments (NAFs)-NAF(56), NAF(20), and NAF(8)-from waxy corn starch with a hydrodynamic diameter of 227, 56, 20, and 8 nm, respectively, were randomly labeled with 1-pyrenebutyric acid. The efficiency of these pyrene-labeled amylopectin-based polysaccharides (Py-AbPS) for pyrene excimer formation (PEF) upon diffusive encounter between an excited and a ground-state pyrene increased with increasing concentration of unlabeled NAF(56) in Py-AbPS dispersions in DMSO. Fluorescence decay analysis of the Py-AbPS dispersions in DMSO prepared with increasing [NAF(56)] yielded the maximum number (Nblobexp) of anhydroglucose units (AGUs) separating two pyrene-labeled AGUs while still allowing PEF. Comparison of Nblobexp with Nblobtheo, obtained by conducting molecular mechanics optimizations on helical oligosaccharide constructs with HyperChem, led to a relationship between the interhelical distance (dh-h) in a cluster of oligosaccharide helices, [NAF(56)], and the number of helices in a cluster. It was found that the AbPSs were composed of building blocks made of 3.5 (±0.9) helices that self-assembled into increasingly larger clusters with increasing [NAF(56)]. The ability of PEF-based experiments to yield the cluster size of AbPSs provides a new experimental means to probe the interior of AbPSs at the molecular level.

Unfolding of Helical Poly(L-Glutamic Acid) in N,N-Dimethylformamide Probed by Pyrene Excimer Fluorescence (PEF).

The denaturation undergone by α-helical poly(L-glutamic acid) (PLGA) in N,N-dimethylformamide upon addition of guanidine hydrochloride (GdHCl) was characterized by comparing the fluorescence of a series of PLGA constructs randomly labeled with the dye pyrene (Py-PLGA) to that of a series of Py-PDLGA samples prepared from a racemic mixture of D,L-glutamic acid. The process of pyrene excimer formation (PEF) was taken advantage of to probe changes in the conformation of α-helical Py-PLGA. Fluorescence Blob Model (FBM) analysis of the fluorescence decays of the Py-PLGA and Py-PDLGA constructs yielded the average number () of glutamic acids located inside a blob, which represented the volume probed by an excited pyrenyl label. remained constant for randomly coiled Py-PDLGA but decreased from ~20 to ~10 glutamic acids for the Py-PLGA samples as GdHCl was added to the solution. The decrease in reflected the decrease in the local density of PLGA as the α-helix unraveled in solution. The changes in with GdHCl concentration was used to determine the change in Gibbs energy required to denature the PLGA α-helix in DMF. The relationship between and the local density of macromolecules can now be applied to characterize the conformation of macromolecules in solution.

Interior of Amylopectin and Nano-Sized Amylopectin Fragments Probed by Viscometry, Dynamic Light Scattering, and Pyrene Excimer Fluorescence.

Nano-sized amylopectin fragments (NAFs), prepared by extrusion of waxy corn starch, were investigated by viscometry, dynamic light scattering (DLS), and pyrene excimer fluorescence (PEF). NAF57, with a hydrodynamic diameter of 57 nm, was treated with nitric acid to yield three degraded NAFs, which appeared to share the same interior and structural features as amylopectin based on their measured intrinsic viscosity and hydrodynamic diameter. This conclusion was further supported by comparing the efficiency of forming excimer between an excited and a ground-state pyrenyl label covalently attached to the NAFs (Py-NAFs) using their IE/IM ratio of the fluorescence intensity of the excimer (IE) to that of the monomer (IM). The overlapping trends obtained for all Py-NAFs and the pyrene-labeled amylopectin samples by plotting the IE/IM ratio as a function of pyrene content provided further evidence that the interior of NAFs and amylopectin shared the same structural features and contained a similar amount of free volume as predicted by the Solution-Cluster (Sol-CL) model. The presence of free volume was validated by adding linear poly(ethylene glycol) (PEG) chains that could not penetrate the interior of Py-NAFs, thus subjecting the Py-NAFs to increased osmotic pressure, which induced their compression and resulted in an increase in IE/IM.

Direct Measure of the Local Concentration of Pyrenyl Groups in Pyrene-Labeled Dendrons Derived from the Rate of Fluorescence Collisional Quenching.

The model-free analysis (MFA) was applied to measure the average rate constant () for pyrene excimer formation (PEF) in a series of pyrene-labeled dendrons referred to as Pyx-G(N), where x (= 2N) is the number of pyrenyl labels born by a dendron of generation N ranging from 1 to 6. was measured in four different solvents, namely tetrahydrofuran (THF), toluene, N,N-dimethylformamide (DMF), and dimethylsulfoxide (DMSO). was found to increase linearly with increasing local pyrene concentration ([Py]loc), where [Py]loc had been determined mathematically for the Pyx-G(N) dendrons. The slope of each straight line changed with the nature of the solvent and represented kdiff, the bimolecular rate constant for PEF. kdiff depended on the solvent viscosity (η) and the probability (p) for PEF upon encounter between an excited and a ground-state pyrene. In a same solvent, kdiff for the Pyx-G(N) dendrons was about 360 ± 30 times smaller than kdiff obtained for ethyl 4-(1-pyrene)butyrate (PyBE), a pyrene model compound similar to the pyrene derivative used to label the dendrons. The massive decrease in kdiff observed for the Pyx-G(N) samples reflected the massive loss in mobility experienced by the pyrenyl labels after being covalently attached onto a macromolecule compared to freely diffusing PyBE. Interestingly, the kdiff values obtained for the Pyx-G(N) dendrons and the PyBE model compound followed similar trends as a function of solvent, indicating that the difference in behavior between the kdiff values obtained in different solvents were merely due to the changes in the η and p values between the solvents. Normalizing the values obtained with the Pyx-G(N) dendrons by the kdiff values obtained for PyBE in the same solvents accounted for changes in η and p, resulting in a master curve upon plotting /(fdiff × kdiff) as a function of [Py]loc, where fdiff was introduced to account for some pyrene aggregation in the higher generation dendron (Py64-G(6)). This result demonstrates that represents a direct measure of [Py]loc in pyrene-labeled macromolecules.

Cyclodextrin supramolecular inclusion-enhanced pyrene excimer switching for highly selective detection of RNase H.

Here, we report a novel fluorescence method for the highly selective and sensitive detection of RNase H by combining the use of a dual-pyrene-labeled DNA/RNA duplex with supramolecular inclusion-enhanced fluorescence. Initially, the probe is in the "off" state due to the rigidness of the double-stranded duplex, which separates the two pyrene units. In the presence of RNase H, the RNA strand of the DNA/RNA duplex will be hydrolyzed, and the DNA strand transforms into a hairpin structure, bringing close the two pyrene units which in turn enter the hydrophobic cavity of a γ-cyclodextrin. As a result, the pyrene excimer emission is greatly enhanced, thereby realizing the detection of RNase H activity. Under optimal conditions, RNase H detection can be achieved in the range from 0.08 to 4 U/mL, with a detection limit of 0.02 U/mL.

Bromide counterion as a spectroscopic sensor at the interface of cetyltrimethylammonium micelles.

The strong UV absorption of the bromide in aqueous solution undergoes a remarkable red shift of more than 10 nm induced by the addition of the salts that constitute a saline buffer. The maximum absorption wavelength of the bromide is displaced from approximately 194 nm in ultrapure water to wavelengths above 200 nm, depending on the composition of the solution. The bromide spectrum as counterion of the cetyltrimethylammonium in the surfactant CTAB also shows sensitivity to the aggregation behavior of the tensioactive, being able to detect intermolecular interactions even at concentrations lower than the critical micelle concentration. And, when the micelles are assembled, the bromide absorption detects the interfacial rearrangements caused by the incorporation of ions. To know more about those interfacial features, the pyrene molecular probe was used, taking advantage of the extensive knowledge of its spectroscopy. Pyrene verifies the existence of changes in the interfacial organization which confirm that the sensitivity of the bromide spectrum is based on the ability of the ion to detect its microenvironment, and therefore reaffirms that its absorption spectrum can be used as a local sensor. The present work encourages the use of bromide as a sensor ion in the UV region between 190 and 210 nm, which would avoid the introduction of external molecular probes that could disturb the system.