Conformational switching of the pyridine-pyrimidine-pyridine scaffold for ion-controlled FRET

Fluorescence resonance energy transfer (FRET) has become a major tool for the static and dynamic study of conformational changes in biological systems. We report herein the investigation of a switchable pyridine-pyrimidine-pyridine scaffold as a support to ion-controlled intramolecular FRET. A dissymmetrical switch bearing naphthalene and acridine fluorophores was synthesized and its photophysical behavior studied. In the neutral state where the molecule adopts a U-shape, the emission of the naphthalene is quenched while a strong emission from the acridine fluorophore is observed, consistent with energy transfer between the naphthalene and the acridine units. The emission of the acridine is also enhanced by the pyridine-induced sensitization (excitation at 280 nm). After introduction of a copper(I) cation which switches the conformation to a W-shape, the complex formed shows the emission of both the naphthalene and acridine units when excited at 280 nm, although coordination also leads to a strong quenching of emission.     

Fluorophore(s) Appended Fullerene Dyads and Triads for Probing Photoinduced Energy Transfer: Syntheses, Electronic Structure, and Fluorescence Studies

Fullerene, C₆₀ was functionalized to possess one or two fluorophore entities. The fluorophore–fullerene dyads thus synthesized contain either a naphthalene, pyrene, or fluorene entity while the triads contain either a pyrene or fluorene entity in addition to a naphthalene entity. The redox behavior of these dyads and triads were probed by cyclic voltammetric technique, while the geometry and electronic structures were deduced from ab initio B3LYP/3-21G(*) method. Steady-state emission studies revealed the occurrence of energy transfer from the singlet excited fluorophore to the fullerene entity in the case of the dyads while the occurrence of step-by-step sequential energy transfer is envisioned in the case of the triads. A better ‘antenna-effect’ owing to the extended range of excitation wavelength to induce energy transfer to the appended fullerene has been achieved in the case of the triads.     

Phenothiazine–rhodamine‐based colorimetric and fluorogenic ‘turn‐on' sensor for Zn2+ and bioimaging studies in live cells

A phenothiazine–rhodamine (PTRH) fluorescent dyad was synthesized and its ability to selectively sense Zn²⁺ ions in solution and in in vitro cell lines was tested using various techniques. When compared with other competing metal ions, the PTRH probe showed the high selectivity for Zn²⁺ ions that was supported by electronic and emission spectral analyses. The emission band at 528 nm for the PTRH probe indicated the ring closed form of PTRH, as for Zn²⁺ ion binding to PTRH, the λ_em get shift to 608 nm was accompanied by a pale yellow to pink colour (under visible light) and green to pinkish red fluorescence emission (under UV light) due to ring opening of the spirolactam moiety in the PTRH ligand. Spectral overlap of the donor emission band and the absorption band of the ring opened form of the acceptor moiety contributed towards the fluorescence resonance energy transfer ON mechanism for Zn²⁺ ion detection. The PTRH sensor had the lowest detection limit for Zn²⁺, found to be 2.89 × 10^?8 M. The sensor also demonstrated good sensing application with minimum toxicity for in vitro analyses using HeLa cells.     

Spectroscopic and functional characterization of an environmentally sensitive fluorescent actin conjugate

Rabbit skeletal muscle F-actin has been selectively labeled at a cysteine residue with the environmentally sensitive fluorophore 6-acryloyl-2-(dimethylamino)naphthalene. The fluorescent actin conjugate behaves similarly to native actin with respect to the polymerization kinetics, critical monomer concentration, and ability to form F-actin paracrystals. Upon polymerization to F-actin, the absorption of the actin conjugate is red-shifted, whereas the fluorescence emission is blue-shifted 740 wavenumbers and is accompanied by a decrease in the fluorescence bandwidth of 470 wavenumbers. These large shifts in the spectral properties of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) in actin provide a simple method for obtaining a spectral discrimination between the G- and F-actin populations during the polymerization reaction. Steady-state fluorescence techniques were used to study the environment of the fluorophore in the monomeric and polymeric forms of actin. Fluorescence emission spectral analysis and quenching and polarization studies of G-actin-Prodan indicated that the fluorophore lies immobile on the protein surface but with one of its faces in full contact with the solvent. In F-actin, the fluorophore has a limited exposure to the solvent and is located in a dielectric environment similar to those seen for Prodan in polar, aprotic solvents or buried within a protein matrix [Macgregor, R. B., Jr., & Weber, G. (1986) Nature (London) 318, 70-73]. Additionally, our results demonstrate that the Prodan molecule conjugated to F-actin is completely immobile during its fluorescence lifetime, exhibits an increase in the resonance energy transfer (RET) from tryptophan residues compared to that observed in G-actin, and shows evidence of homologous RET within the polymer.     

Exploring aggregation‐induced emission through tuning of ligand structure for picomolar detection of pyrene

Tuning of ligand structures through controlled variation of ring number in fused‐ring aromatic moiety appended to antipyrine allows detection of 7.8 × 10^?12 M pyrene via aggregation‐induced emission (AIE) associated with 101‐fold fluorescence enhancement. In one case, antipyrine unit is replaced by pyridine to derive bis‐methylanthracenyl picolyl amine. The structures of four molecules have been confirmed by single crystal X‐ray diffraction analysis. Among them, pyrene‐antipyrine conjugate (L) undergoes pyrene triggered inhibition of photo‐induced electron transfer (PET) leading to water‐assisted AIE.     

A novel homogeneous bioluminescence resonance energy transfer element for biomolecular detection with CCD camera or CMOS device

A novel optical signal element based on homogeneous bioluminescence resonance energy transfer (BRET) was developed for biomolecular detection. A fluorescent dye and alkaline phosphatase (AP) conjugate was used as a reporter and light‐generation element for imaging detection platforms that use a CCD camera or CMOS chip‐based devices. In the presence of a luminescence substrate, the energy from the first light emission of a bioluminescence enzymatic reaction was transferred to fluorescent dyes which were conjugated to an enzyme. This resulted in a second light emission with a shorter wavelength. The second light was localized at the position of target molecules without the diffusion problems present in current technology. To optimize energy transfer efficiency, the ratio of enzyme to fluorophore in the conjugates, the fluorescent dyes used in the conjugates and the luminescence substrates used for BRET were investigated. BRET was demonstrated by using both a CCD camera and a CMOS imaging device. Image spatial resolution was greatly improved compared with conventional chemiluminescence detection. This new signal element opens a door for the direct measurement of fluorescent signals on an imaging chip without an external light source and portable instrumentation normally required for the fluorescent detection of biomolecules. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis of a ratiometric fluorescent peptide sensor for the highly selective detection of Cd2+

A novel ratiometric fluorescent peptidyl chemosensor (Dansyl-Cys-Pro-Gly-Cys-Trp-NH(2), D-P5) for metal ions detection has been synthesized via Fmoc solid-phase peptide synthesis. The chemosensor exhibited a high selectivity for Cd(2+) over other metal ions including competitive transition and Group I and II metal ions in neutral pH. The fluorescence emission intensity of D-P5 was significantly enhanced in the presence of Cd(2+) by fluorescent resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF) effects. The binding stoichiometry, detection limit, binding affinity, reversibility and pH sensitivity of the sensor for Cd(2+) were investigated.     

Fluorescence resonance energy transfer in a novel cyclodextrin-peptide conjugate for detecting steroid molecules

A novel cyclodextrin conjugated peptide, 1, having two different fluorophores, coumarin and pyrene, in the side chains has been designed and synthesized. The circular dichroism study reveals that 1 shows typical -helix pattern, and forms intramolecular inclusion complex with coumarin. The fluorescence emission study shows that the peptide exhibits intramolecular fluorescence resonance energy transfer (FRET) without quenching of two fluorophores. We have determined the binding constants of 1 for various biologically important steroid molecules as guests using the guest-responsive variation in the fluorescence emission intensity of coumarin.     

Pyrene binary probes for unambiguous detection of mRNA using time-resolved fluorescence spectroscopy

We report here the design, synthesis and application of pyrene binary oligonucleotide probes for selective detection of cellular mRNA. The detection strategy is based on the formation of a fluorescent excimer when two pyrene groups are brought into close proximity upon hybridization of the probes with the target mRNA. The pyrene excimer has a long fluorescence lifetime (>40 ns) compared with that of cellular extracts (~7 ns), allowing selective detection of the excimer using time-resolved emission spectra (TRES). Optimized probes were used to target a specific region of sensorin mRNA yielding a strong excimer emission peak at 485 nm in the presence of the target and no excimer emission in the absence of the target in buffer solution. While direct fluorescence measurement of neuronal extracts showed a strong fluorescent background, obscuring the detection of the excimer signal, time-resolved emission measurements indicated that the emission decay of the cellular extracts is ~8 times faster than that of the pyrene excimer probes. Thus, using TRES of the pyrene probes, we are able to selectively detect mRNA in the presence of cellular extracts, demonstrating the potential for application of pyrene excimer probes for imaging mRNAs in cellular environments that have background fluorescence.     

Proximity of lectin receptors on the cell surface measured by fluorescence energy transfer in a flow system.

Molecules of the lectin concanavalin A have been labeled separately with the fluorescein and rhodamine chromophores and jointly bound to the surface of transformed Friend erythroleukemia cells. The two dyes constitute an ideal donor-acceptor pair for fluorescence resonance energy transfer thereby permitting the determination of the proximity relationships between bound ligand molecules and the corresponding surface receptors. The transfer efficiency at saturation (about 57%) was measured in a multiparameter flow system using laser excitation at 488 nm and detection of fluorescein and rhodamine emission intensities as well as the emission anisotropy of the rhodamine fluorescence for each cell. The degree of energy transfer was estimated from the quenching of donor emission, the sensitization of acceptor emission, and the depolarization of acceptor fluorescence. The system has been modeled according to a formalism developed by Gennis and Cantor (Biochemistry 11: 2509, 1972). We estimate the separation between the surfaces of bound lectin molecules at saturation to be 0-40 A, a range possibly characteristic for micropatches induced by ligand binding.     

An azo-based PNA monomer: synthesis and spectroscopic study

The full synthetic details and photospectroscopic characterization of a peptide nucleic acid (PNA) monomer suitable for Fmoc-based oligomerization chemistry that bears an azobenzene moiety as a base surrogate are reported. The monomer showed the ability to quench the fluorescence emission of fluorescein and pyrene luminophores and proved to be a competent F?ster resonance energy transfer partner in a PNA-based molecular beacon.     

Surface Plasmon Tunability and Emission Sensitivity of Ultrasmall Fluorescent Copper Nanoclusters

Ultrasmall copper nanoparticles have been synthesized using copper(II) salt as precursor by hydrazine reduction in the presence of citric acid and cetyltrimethylammonium bromide facilitating the growth of stable copper nanoparticles with an average diameter of <2 nm. The corresponding surface plasmon resonances were monitored under variable microenvironments, and it is seen that these tiny copper nanoparticles form aggregates under stipulated reaction conditions. It is noted that ultrasmall copper nanoparticles do not exhibit any characteristic surface plasmon band in the visible region; rather, a continuous absorption is seen over the entire UV–vis region. However, a well-defined plasmon absorption band makes its appearance while the particles are aggregated in close-packed assembly. These results demonstrate that the maximum of surface plasmon resonance is red-shifted from that of isolated particles because of electromagnetic interaction between the particles. The aggregation process is manifested upon changes of pH, anionic surfactant, etc. and is not reversible, i.e., the aggregates could not be re-dispersed into ultrasmall particles. The effect of addition of electrolyte has been monitored to study the surface plasmon damping of the copper nanoparticles. The plasmonic sensitivity of the copper nanoparticle aggregates has been elicited by the determination of amino acid chain length with exquisite sensitivity because of enormous electromagnetic field at the junction of the particles in the aggregates. Interestingly, the as-synthesized ultrasmall copper nanoclusters exhibit excellent fluorescence properties with a narrow emission profile. The emission properties of these copper nanoclusters have been utilized as an indicator for selective and ultrasensitive detection of highly toxic Hg^II ions in water in the nanomolar detection limit.     

Simultaneous determination of naphthalene and anthraquinone derivatives in Rumex nepalensis Spreng. roots by HPLC: comparison of different extraction methods and validation

Introduction – Rumex nepalensis contains mainly anthraquinone and naphthalene derivatives. Although HPLC methods have been reported for the analysis of anthraquinones, neither a phytochemical analysis of Rumex species nor the simultaneous determination of anthraquinone and naphthalene derivatives in other samples has been reported so far. Objective – To develop and validate a HPLC method for the simultaneous determination of anthraquinone and naphthalene derivatives in R. nepalensis roots. Methodology – Anthraquinones and naphthalenes were extracted from R. nepalensis roots by three methods (reflux, ultrasonication and pressurized liquid extraction) using methanol. Separation was achieved on an RP C₁₈ column with a gradient mobile phase consisting of 0.05% orthophosphoric acid in water (solvent A) and methanol (solvent B) using a UV detector (254 nm). Results – Small differences were observed in the contents of anthraquinone and naphthalene derivatives extracted by the three methods. Chrysophanol‐8‐O‐β‐D‐glucopyranoside and nepodin were detected as major constituents. The method showed a good linearity (r² > 0.9992), high precision (RSD < 5%) and a good recovery (97–105%) of the compounds. The lowest detection limit was found to be 0.97 ng and the method was found to be robust. Conclusion – Reflux and ultrasonication were found to be the best suited methods for the extraction of glycosides and aglycones, respectively. The developed and validated HPLC method is simple, precise and accurate; and can hence be recommended as the method of choice for the analysis of anthraquinones and naphthalenes in R. nepalensis and other Rumex species for both quality control as well as routine analytical purposes. Copyright © 2010 John Wiley & Sons, Ltd.     

A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes

A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl-labeled lipids; energy donors and perylenoyl-labeled lipids; energy acceptors) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl-triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The nonpolar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function.     

Investigation on the analytical application of cationic Gemini surfactant 12‐4‐12 and its interaction with DNA

The quantitative determination of nucleic acids is of great importance in fundamental research and clinical diagnosis. In this work, the interaction between DNA and cationic Gemini surfactant 12‐4‐12, which changes the conformation of DNA, was investigated by UV‐vis absorption, FT‐IR spectra and steady‐state fluorescence techniques. A hydrophobic pyrene probe was used to investigate the microenvironment change and calculate the critical micelle concentration (CMC) of Gemini surfactant 12‐4‐12 (0.69 mmol/L), which is close to the value obtained from the conductivity method (0.79 mmol/L). A new detection assay for DNA is proposed with Gemini surfactant 12‐4‐12, using the resonance light‐scattering (RLS) technique. The formation of DNA–12‐4‐12 complex resulted in enhanced RLS signals at 368 nm, which is proportional to DNA concentration in the range 0.304–5.32 mg/L, with a detection limit of 35 µg/L. Most coexisting substances do not interfere in the detection and four synthetic samples were analyzed satisfactorily. Copyright © 2011 John Wiley & Sons, Ltd.     

High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy

Detection of Forster resonance energy transfer (FRET) between fluorescent protein labeled targets is a valuable strategy for measurement of protein-protein interactions and other intracellular processes. Despite the utility of FRET, widespread application of this technique to biological problems and high-throughput screening has been limited by low-contrast measurement strategies that rely on the detection of sensitized emission or photodestruction of the sample. Here we report a FRET detection strategy based on detecting depolarized sensitized emission. In the absence of FRET, we show that fluorescence emission from a donor fluorescent protein is highly polarized. Depolarization of fluorescence emission is observed only in the presence of energy transfer. A simple detection strategy was adapted for fluorescence microscopy using both laser scanning and wide-field approaches. This approach is able to distinguish FRET between linked and unlinked Cerulean and Venus fluorescent proteins in living cells with a larger dynamic range than other approaches.     

Application of a lanthanide composite nanoparticle‐sensitized luminescence method for the determination of salicylic acid in pharmaceutical formulations and human plasma

Terbium‐acetylacetone (Tb–acac) composite nanoparticles were synthesized using the ultrasonic method. The nanoparticles are water‐soluble, stable and have extremely narrow emission bands and high internal quantum efficiencies. They were used as fluorimetric probes in the determination of salicylic acid (SA), based on the fluorescence enhancement of nanoparticles through fluorescence resonance energy transfer (FRET). The influence of buffer solution was investigated. Under the optimum conditions, a linear calibration graph was obtained over the SA concentration range 5 × 10^–7–1 × 10^–4 mol/L. The limit of detection was found to be 2.5 × 10^–8 mol/L. The relative standard deviation (RSD) for six repeated measurements of 1 × 10^–4 mol/LSA was 1.75%. The method was applied to the determination of SA in pharmaceutical formulations and human plasma. We believe that the proposed approach has great potential for clinical purposes. Copyright © 2008 John Wiley & Sons, Ltd.     

Detection of benzo(a)pyrene photodegradation products using DNA electrochemical sensors

The reactivity of photodegradation products of benzo(a)pyrene vs. DNA has been assessed using both genomic and oligonucleotide based DNA electrochemical sensors. The kinetic of a photooxidation reaction of benzo(a)pyrene (BaP) carried out in controlled conditions using a 6 W UV lamp peaked at 365 nm has been studied using LC with fluorimetric detection. Degradation of benzo(a)pyrene by both UV and UV/H(2)O(2) exhibited pseudo-first-order reaction kinetics with half-lives ranging from 3.0 to 9.8h depending on the pH and on the amount of H(2)O(2). The oxidation products of benzo(a)pyrene obtained in different conditions were tested on genomic ssDNA electrochemical sensors obtained via immobilisation of salmon testis ss-DNA on graphite screen-printed electrodes. Guanines oxidation signals obtained using chronopotentiometry were used to detect the interaction of the products with DNA. The dose-response curve obtained with benzo(a)pyrene incubated 24 h at pH 4.7 was different from that of the parent compound indicating a different type of interaction with DNA. A DNA hybridisation sensor was also assembled using a thiolated/biotynilated 24-mer oligonucleotide immobilised on a gold screen-printed electrode and avidin-alkaline phosphatase conjugate. A voltammetric detection of naphtol was used to detect the hybridisation reaction. A net inhibition of the hybridisation reaction was observed after incubation with benzo(a)pyrene oxidation products that was attributed to the formation of stable adducts with the guanines of the biotinylated strand. LC-MS-MS studies of the oxidation products confirmed the presence of chemical species potentially forming adducts with DNA. The data reported demonstrate that DNA electrochemical sensors have the potential to be used to monitor remediation processes and to assess the potential toxicity vs. DNA of chemicals forming stable DNA adducts.     

The ensemble of hetero-proteins in inorganic nanochannels

The assembly and proper alignment of two heterofluorescent proteins (sGFP and DsRed) in the mesoporous channels of ethanol-treated FSM6.2 (a folded-sheet mesoporous material with a pore diameter of 6.2 nm) was confirmed using a fluorescence resonance energy transfer (FRET) technique. The sGFP-DsRed-FSM6.2 conjugate showed a large decrease in the emission of donor (sGFP) fluorescence, indicating that the conjugate functions as an energy transfer system through the combination of the two heteroproteins, due to the successful encapsulation of the sGFP-DsRed pairs in the mesopores. Fluorescence spectral analysis demonstrated that the proteins were highly dispersed and homogeneously encapsulated in the mesopores of FSM6.2, even at high concentration, although they spontaneously aggregated and showed a red shift in solution at the concentration corresponding to that in the conjugate. Furthermore, an increase in the amount of sGFP and DsRed adsorbed to the pores of FSM6.2 led to a decrease in the distance between these proteins, resulting in enhancement of FRET efficiency.     

Two‐channel near‐infrared fluorescence Ag+ ion sensing of a new star‐shaped dendrimer

A new near‐infrared fluorescence sensor PDI‐PD for Ag⁺ ions was successfully prepared and its structure characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR and high‐resolution mass spectrometry; matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (HRMS MALDI‐TOF). The probe exhibited rapid, sensitive, and selective two‐channel fluorescence responses towards Ag⁺ ions and protons. The probe has a marked high binding affinity and high sensitivity for Ag⁺, with a detection limit of 1.4 × 10^?6 M. An approximately five‐fold enhanced core emission at 784 nm was attributed to fluorescence resonance energy transfer (FRET). The enhanced core emission of the probe with Ag⁺ ions based on photo‐induced electron transfer and FRET is discussed. In addition, the probe presented a visible colour change. All experimental results demonstrated that PDI‐PD is an efficient tool for the selective, sensitive and rapid detection of Ag⁺ ions and protons using two‐channel fluorescence responses.