Fluorescent Chromophore Research Articles

Optical mapping of nonmelanoma skin Cancers-A pilot clinical study.

Nonmelanoma skin cancer (NMSC) is the most common human cancer. Because these tumors often affect the face, there is a strong need for both accurate removal of these neoplasms to prevent recurrence and maximal tissue preservation to prevent cosmetic or functional deformity. Polarization-enhanced reflectance and fluorescence imaging (PERFI) is a new bedside method that uses fluorescent chromophores to image NMSC. While the feasibility of the technique has been successfully demonstrated in ex vivo studies, this is the first pilot study to extend the use of PERFI to in vivo intraoperative imaging of NMSC. Subjects were recruited from a population of patients with biopsy-confirmed NMSC, scheduled for Mohs micrographic surgery. Eight cases were studied. Sterile methylene blue (MB) was diluted in anaesthetic solution and infused into the peritumoral space. Digital photographs of the lesion were taken and Mohs surgery was performed. Then, the surgical bed was re-imaged. Each excision was also imaged ex vivo and processed for routine histopathology. Optical images were processed and compared with histopathology. The injection of MB was well tolerated. We observed a transient blue staining of the treated area, which disappeared completely within 1 week in all of the patients. In all subjects, the contrast agent, MB, was preferentially retained in the tumor. The ex vivo images correlated well with histopathology. In vivo images qualitatively delineated the tumor margins. The results of our pilot trial indicate that PERFI may be useful for accurate and rapid delineation of NMSC during surgery. Lasers Surg. Med. 49:803-809, 2017. © 2017 Wiley Periodicals, Inc.

Combined quantum-mechanical molecular mechanics calculations with NWChem and AMBER: Excited state properties of green fluorescent protein chromophore analogue in aqueous solution.

Combined quantum mechanical molecular mechanics (QM/MM) calculations have become a popular methodology for efficient and accurate description of large molecular systems. In this work we introduce our development of a QM/MM framework based on two well-known codes-NWChem and AMBER. As an initial application area we are focused on excited state properties of small molecules in an aqueous phase using an analogue of the green fluorescent protein (GFP) chromophore as a particular test case. Our approach incorporates high level coupled cluster theory for the analysis of excited states providing a reliable theoretical analysis of effects of an aqueous solvation environment on the photochemical properties of the GFP chromophore. Using a systematic approach, which involves comparison of gas phase and aqueous phase results for different protonation states and conformations, we resolve existing uncertainties regarding the theoretical interpretation of experimental data. We observe that the impact of aqueous environment on charged states generally results in blue shifts of the absorption spectra, but the magnitude of the effect is sensitive to both protonation state and conformation and can be rationalized based on charge movement into the area of higher/lower external electrostatic potentials. At neutral pH levels the experimentally observed absorption signal is most likely coming from the phenol protonated form. Our results also show that the high level electron correlated method is essential for a proper description of excited states of GFP. © 2017 Wiley Periodicals, Inc.

Over 10% EQE Near‐Infrared Electroluminescence Based on a Thermally Activated Delayed Fluorescence Emitter

Significant effort has been made to develop novel material systems to improve the efficiency of near‐infrared organic light‐emitting diodes (NIR OLEDs). Of those, fluorescent chromophores are mostly studied because of their advantages in cost and tunability. However, it is still rare for fluorescent NIR emitters to present good color purities in the NIR range and to have high external quantum efficiency (EQE). Here, a wedge‐shaped D‐π‐A‐π‐D emitter APDC‐DTPA with thermally activated delayed fluorescence property and a small single‐triplet splitting (ΔEst) of 0.14 eV is presented. The non‐doped NIR device exhibits excellent performance with a maximum EQE of 2.19% and a peak wavelength of 777 nm. Remarkably, when 10 wt% of APDC‐DTPA is doped in 1,3,5‐tris(1‐phenyl‐1H‐benzimidazol‐2‐yl)benzene host, an extremely high EQE of 10.19% with an emission peak of 693 nm is achieved. All these values represent the best result for NIR OLEDs based on a pure organic fluorescent emitter with similar device structure and color gamut.

Fluorescence enhancement of organic chromophore in solid state via organic–inorganic hybrid matrix: Steady state and kinetic studies

Xerogels of silica-based organic–inorganic hybrid materials consisting of covalently bound fluorophores, terthiophene and terphenylene derivatives, were fabricated and analyzed. The synthesized chromophore-containing alkoxysilane derivatives were no emission in solid state, but showed strong fluorescence in organic–inorganic hybrid materials as well as in solution. Moreover, the fluorescence quantum yield of chromophore could be further improved in organic–inorganic hybrid materials with the optimized component than that of chromophore dissolved in solution. On the other hand, the fluorescence intensity of hybrid materials decreased with increasing the concentration of chromophore in organic–inorganic hybrid materials. Obviously, the relative distance between chromophores was shortened to perturb radiative relaxation. Temperature-dependent fluorescence spectra revealed that the fluorescence of chromophore in organic–inorganic hybrid materials was temperature-independent. This result indicated the inter-chromophore interactions and intra-molecular motion have been forbidden. The kinetic measurements confirmed that the chromophore spread in organic–inorganic hybrid materials was fixed with rigid conformation. The florescence decay lifetime of chromophore also kept similar at different temperature with organic–inorganic hybrid materials skeleton. This hybrid matrix provided a platform that enhanced fluorescence of chromophore in solid state at ambient temperature. Emission enhancement of organic chromophores, terthiophene and terphenylene, was performed through the utilization of covalently bound silica-based organic–inorganic hybrid material (OIHM). The chromophore was no emission in solid state, but showed strong fluorescence in OIHM as well as in solution. Moreover, the fluorescence quantum yield of chromophore could be better in OIHM with optimized component than that in solution. Temperature-dependent fluorescence spectra revealed that the fluorescence of chromophore in OIHM was temperature-independent. The kinetic measurements confirmed that the chromophore spread in OIHM was fixed with rigid conformation. This OIHM system offered a useful platform to maintain the emission behavior of organic chromophores in quasi-solid state.

Chemosensing hybrid materials: Chalcones‐functionalized cage‐like mesoporous SBA‐16 material for highly selective detection of toxic metal ions

Highly selective and low‐cost optical nanosensors of organic–inorganic hybrid materials for heavy metal ions detection have been prepared via the functionalization of mesoporous silica (SBA‐16) with chalcone fluorescent chromophores. The successful attachment of organic chalcone moieties and preservation of original structure of SBA‐16 after the anchoring process were confirmed by extensive characterizations using various techniques like Fourier transform infrared and UV–visible spectroscopies, transmission electron microscopy, nitrogen adsorption–desorption isotherms, low‐angle X‐ray diffraction and thermogravimetric analysis. The colorimetric behaviour, selectivity and sensitivity were also investigated. The optical nanosensors respond selectively to heavy metal ions, such as Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+, with observable colour changes in 0.01 M Tris–HCl aqueous buffer solution. Also, the optical sensing ability of the investigated nanosensors to the mentioned metal ions was investigated using steady‐state absorption and emission techniques. Significant increase in the absorption spectra and a static quenching in the emission spectra are observed upon adding various concentrations of the studied metal ions. The spectral changes as well as the observable colour changes suggest that the investigated nanosensors are suitable for simple, economic, online analysis and remote design of these toxic metal ions with fast kinetic responses. Finally, the low detection limits for all the studied metals are in good agreement with those recommended by both the US Environmental Protection Agency and World Health Organization, except for Hg2+ and Cd2+, indicating that the investigated nanosensors have hypersensitivity, selectivity and better recognition for all the studied metal ions.

Simulation of the UV/Visible Absorption Spectra of Fluorescent Protein Chromophore Models

AbstractTime‐dependent density functional theory has been used to simulate the UV/Vis absorption spectra of fluorescent protein model chromophores with particular focus on the vibronic structure of the lowest‐energy absorption band as a structural fingerprint. Combining the B3LYP‐35 XC functional with non‐equilibrium solvation and the gradient approach provides a vibronic structure close to that recorded experimentally (when comparisons with experiment are possible). This study focuses on the chromophore present in green fluorescent proteins (FP1), in its neutral and deprotonated forms as well as for both cis (Z) and trans (E) conformations. The method was then employed to simulate the spectra of structures for which experimental spectra are not available or not well resolved. The effect of extending the conjugation of the chromophore, by adding unsaturated moieties such as in the chromophore of DsRed (FP2), was considered. Overall, among these three investigated structural modifications (neutral versus anionic, smaller versus larger chromophore, and cis versus trans conformations), absorption spectra can only distinguish unambiguously between the neutral and anionic forms. An extension of the π‐conjugation length of the chromophore is, as would be expected, reflected in a bathochromic shift of the absorption band maximum, but modifications of the chromophore environment in proteins can also induce shifts of the absorption maximum, and they are difficult to distinguish from each other.

Iron Release from the Siderophore Pyoverdine in Pseudomonas aeruginosa Involves Three New Actors: FpvC, FpvG, and FpvH.

Siderophores are iron chelators produced by bacteria to access iron, an essential nutriment. Pyoverdine (PVDI), the major siderophore produced by Pseudomonas aeruginosa PAO1, consists of a fluorescent chromophore linked to an octapeptide. The ferric form of PVDI is transported from the extracellular environment into the periplasm by the outer membrane transporter, FpvA. Iron is then released from the siderophore in the periplasm by a mechanism that does not involve chemical modification of the chelator but an iron reduction step. Here, we followed the kinetics of iron release from PVDI, in vitro and in living cells, by monitoring its fluorescence (as apo PVDI is fluorescent, whereas PVDI-Fe(III) is not). Deletion of the inner membrane proteins fpvG (PA2403) and fpvH (PA2404) affected 55Fe uptake via PVDI and completely abolished PVDI-Fe dissociation, indicating that these two proteins are involved in iron acquisition via this siderophore. PVDI-Fe dissociation studies, using an in vitro assay, showed that iron release from this siderophore requires the presence of an iron reducer (DTT) and an iron chelator (ferrozine). In this assay, DTT could be replaced by the inner membrane protein, FpvG, and ferrozine by the periplasmic protein, FpvC, suggesting that FpvG acts as a reductase and FpvC as an Fe2+ chelator in the process of PVDI-Fe dissociation in the periplasm of P. aeruginosa cells. This mechanism of iron release from PVDI is atypical among Gram-negative bacteria but seems to be conserved among Pseudomonads.

A New Class of Lasing Materials: Intrinsic Stimulated Emission from Nonlinear Optically Active Metal–Organic Frameworks

Blue-color stimulated emission with low threshold power is observed from In- and Zn-MOFs, which feature a highly fluorescent chromophore densely packed and rigidly linked to the metal-ion centers in the solid state. The density-of-states and transition dipole moments are calculated and the stimulated emission phenomenon is correlated with these properties.

The Effect of Conjugation on the Competition between Internal Conversion and Electron Detachment: A Comparison between Green Fluorescent and Red Kaede Protein Chromophores.

Kaede, an analogue of green fluorescent protein (GFP), is a green-to-red photoconvertible fluorescent protein used as an in vivo "optical highlighter" in bioimaging. The fluorescence quantum yield of the red Kaede protein is lower than that of GFP, suggesting that increasing the conjugation modifies the electronic relaxation pathway. Using a combination of anion photoelectron spectroscopy and electronic structure calculations, we find that the isolated red Kaede protein chromophore in the gas phase is deprotonated at the imidazole ring, unlike the GFP chromophore that is deprotonated at the phenol ring. We find evidence of an efficient electronic relaxation pathway from higher-lying electronically excited states to the S1 state of the red Kaede chromophore that is not accessible in the GFP chromophore. Rapid autodetachment from high-lying vibrational states of S1 is found to compete efficiently with internal conversion to the ground electronic state.

Mispacking and the Fitness Landscape of the Green Fluorescent Protein Chromophore Milieu.

The autocatalytic maturation of the chromophore in green fluorescent protein (GFP) was thought to require the precise positioning of the side chains surrounding it in the core of the protein, many of which are strongly conserved among homologous fluorescent proteins. In this study, we screened for green fluorescence in an exhaustive set of point mutations of seven residues that make up the chromophore microenvironment, excluding R96 and E222 because mutations at these positions have been previously characterized. Contrary to expectations, nearly all amino acids were tolerated at all seven positions. Only four point mutations knocked out fluorescence entirely. However, chromophore maturation was found to be slower and/or fluorescence reduced in several cases. Selected combinations of mutations showed nonadditive effects, including cooperativity and rescue. The results provide guidelines for the computational engineering of GFPs.

Emission enhancement and application of synthetic green fluorescent protein chromophore analogs

Emission enhancement and application of GFPc analogs are achieved via chemical modification or physical encapsulation due to conformational motion inhibition.

Fluorescence enhancement of oligodeoxynucleotides modified with green fluorescent protein chromophore mimics upon triplex formation.

Green fluorescent protein (GFP)-based molecular-rotor chromophores were attached to the 5-positions of deoxyuridines, and subsequently, incorporated into the middle positions of oligodeoxynucleotides. These oligonucleotides were designed to form triplex DNA in order to encapsulate the GFP chromophores, mimicking GFP structures. Upon triplex formation, the embedded GFP chromophores exhibited fluorescence enhancement, suggesting the potential application of these fluorescent probes for the detection of nucleic acids.

First observation of 3D aggregates in a single-component Langmuir film below the equilibrium spreading pressure

Single component monolayers from Dipalamitoyl Phosphatidyl Ethanolamine head labelled with the fluorescent chromophore NitroBenzoxaDiazole (DP-NBD-PE) were investigated at the air-water interface as Langmuir films and deposited on silicon wafers or glass plates as Langmuir-Blodgett (LB) films. A step compression and monitoring of the pressure relaxation together with domain formation as observed with Brewster angle microscopy suggests that main transition from liquid expanded to liquid compressed state start at around 6.9 mN/m though on the isotherm it starts around 9 mN/m at 25° C. Brewster angle microscopy also reveals a nonuniform structure in the monolayer. 3D aggregates - cylinders with 50 – 150 nm diameter and bilayer height were observed with Atomic Force Microscopy when deposition was carried at 7 mN/m, above the main phase transition but considerably lower than the equilibrium spreading pressure of 19.6 mN/m for this molecule. When LB film deposition is carried just below the main phase transition a uniform height layer of film in a liquid phase is observed with AFM with no structures. Both compression and deposition were carried at very low speeds with large time to relax in ordered to avoid kinetic effects. These 3D aggregates are not due to the transfer process or interaction with the substrate. These aggregates provide a highly developed area combined with monolayer thick structure which can produce very fast and highly sensitive biosensors.

Ultrasonication Extraction - HPLC-VIS Fluorescence Method Validation for Trace Analysis of Oil and Grease in Water

A new method was developed to determine the oil and grease in water by HPLC-Vis fluorescence technique. Ultrasonication liquid-liquid extraction method was applied by using 1 ml of toluene to 100 ml of water. The analysis was carried out on a LC-10AT-VP chromatograph with a fluorescence RF. 10 a syknm detector equipped with a varian CN (250 × 4.6 μm) Column. The separation was preformed isocratically using a mixture of 90% solvent (n-Hexane) with 10% solvent (Toluene). Asphaltenic compounds were established to be the main fluorescence chromophores (excitation wave length at 420 nm and emission wave length at 500 nm). The correlation coefficient was (0.992), recovery ranged from 92% to 108% and the limit of detection was found to be 0.06 μg.L-1. The method can be utilized successfully for determination of oil and grease in water samples.

Induction of Site-Specific Oxidative Damage at Telomeres by Killerred-Fused Shelretin Proteins.

Chronic oxidative stress is the major endogenous metabolic stress and contributes directly to telomere shortening and senescence. Understanding the dysfunction of telomeres under oxidative stress will greatly facilitate healthy aging and advance the treatment of aging-related diseases. Here, we describe the KR-TEL (KillerRed induced DNA damage at telomeres) system that induces site-specific oxidative damage at telomeres. We have developed the KR-TEL system by fusing killerred with the shelterin component TRF1 (KR-TRF1) or other shelterin proteins. Killerred (KR), an engineered red fluorescent chromophore, is capable of generating site-specific superoxide upon green light activation (550-580 nm). When KR-TRF1 expressing cells are exposed to green or laser light at defined wavelength to activate KR, localized oxidative DNA damage will be induced at telomeres. KR-induced oxidative DNA damage shows a high degree of resemblance to the complex spectrum of DNA damage induced by radiation in terms of the ratios of oxidized bases and DNA strand breaks. Unlike current oxidation-inducing methods (e.g., IR, chemical, and toxicants), which create damage throughout the genome, KR produces spatially limited oxidative DNA damage only in its immediate proximity. This property of KR allows us to engineer oxidative damage specifically at the telomere in a light dose-dependent manner. Using the KR-TEL system to determine the DNA damage response and repair mechanisms at telomeres has several advantages, which make it an ideal system to investigate the mechanism of how telomere integrity is maintained and how this mechanism plays a role in cancer biology.

Preparation of a white-light-emitting fluorescent supramolecular polymer gel with a single chromophore and use of the gel to fabricate a protected quick response code

We report here a novel yet facile approach to prepare white fluorescent polymeric materials based on the aggregation of a single fluorescent chromophore.

The study of metal binding properties and electronic transitions of dithienopyrole derivatives

Fluorescence chromophore (L) based on dithienopyrole derivatives (DPT) have been compared the binding site for detecting metal ions. There are consisting of methacryloyl amine (L1) and cyanoacrylic acid (L2) acceptor, respectively. Corresponding molecular geometries, orbital energies, electron contributions and absorption properties of the fluorescence sensor were investigated by using density functional theory (DFT) with B3LYP method of calculation. The DFT based studies with various solvation and absorption spectra were performed using time-dependent density functional theory (TD-DFT) with the polarized continuum model. Comparing with L2, the experimental L1 shows a red shift both absorption and emission spectra at 350 and 485 nm, respectively. The solvent environment exhibited slight shift in experimental and calculated absorption. An effect of various metal ions on absorption behavior was studied. Results indicate that the L1 and L2 exhibit excellent sensitivity for Pb2+ ion. The binding complex M-L1 and M-L2 (M: Cd, Cu, Hg, Pb, Zn) exhibited Pb<Cu<Zn<Mn<Cd<Hg.

Multicolor imaging of hydrogen peroxide level in living and apoptotic cells by a single fluorescent probe

To understand the entangled relationship between reactive oxygen species (ROS) and apoptosis, there is urgent need for simultaneous dynamic monitoring of these two important biological events. In this study, we have developed a fluorescent probe, pep4-NP1, which can simultaneously detect H2O2 and caspase 3, the respective markers of ROS and apoptosis. The probe contains a H2O2 fluorescence reporter (NP1) and Cy5 fluorescent chromophore connected by a caspase 3 specific recognition peptide. The detecting strategy was realized through a controllable fluorescence resonance energy transfer (FRET) process between NP1 and Cy5 of pep4-NP1, after reaction with H2O2, which was verified by molecular calculation and in vitro spectral studies. In the absent of caspase 3, the accumulation of H2O2 induces red fluorescence of pep4-NP1 centered at 663nm in living cells due to the existence of FRET. In contrast, FRET is inhibited in apoptotic cells due to cleavage of the peptide spacer of pep4-NP1 by over-expressed caspase 3. Consequently, green fluorescence (555nm) predominated when labelling production of H2O2 in apoptotic cells. Moreover, Pep4-NP1 shows excellent selectivity towards H2O2 and caspase 3 on their respective reaction sites. Therefore, pep4-NP1 can distinguish endogenously generated H2O2 between living cells and apoptotic cells with different fluorescence wavelengths, providing additional information on the ROS production pathways.

A novel functionalized nanoporous SBA-15 as a selective fluorescent sensor for the detection of multianalytes (Fe3+ and Cr2O72−) in water

A novel silica-based chemosensor (SBA-Is-INH) was prepared through the functionalization of mesoporous silica SBA-15 material with a fluorescent chromophore, (Z)-N'-(2-oxoindolin-3-ylidene)isonicotinohydrazide. The synthesized materials were characterized by different techniques such as low angle XRD, N2 adsorption-desorption, TGA, TEM and FT-IR spectroscopy. The data showed that the organic moiety (0.41mmolg−1) was successfully grafted to the SBA-15 and the primary hexagonally ordered mesoporous structure of SBA-15 was preserved after the grafting procedure. Photoluminescence spectroscopy has been used to investigate the sensing behavior of this material. SBA-Is-INH showed high selectivity for Fe3+ and Cr2O72− in water over a wide range of tested ions. Upon introducing trace amounts of Fe3+ and Cr2O72− ions into aqueous solution, remarkable quenching in the fluorescence was observed. Furthermore, a good linearity was observed between the fluorescence intensity of SBA-Is-INH and the concentration of Fe3+ and Cr2O72− with satisfactory detection limits of 6.04×10−7M and 5.09×10−7M, respectively. Finally, the proposed method was successfully utilized for the determination of Fe3+ and Cr2O72− in river water, well water and tap water samples.

Green fluorescent protein chromophore derivative suppresses ultraviolet A-induced JNK-signalling and apoptosis in keratinocytes and adverse effects in zebrafish embryos.

Solar ultraviolet (UV) light has been recognized as the important environmental hazard and contributes to diverse skin damage such as cell death, photoageing and even carcinogenesis. Revelation of harmful responses attributed to UVA radiation has promoted the development of photoprotective agents against UVA-induced skin damage. In the present study, we tried to evaluate the potential protective effects of a synthetic green fluorescent protein (GFP) chromophore derivative, 4-chlorobenzyldene-1, 2-dimethylimidazolinone (Cl-BDI, called TC-22) on UVA- and UVB- induced stress responses in skin. The HaCaT keratinocytes were used to evaluate the cellular effects. Zebrafish (Danio rerio), which is regarded as a useful and cost-effective alternative to some mammalian models, was applied as the in vivo animal model. In HaCaT keratinocytes, TC-22 was able to obviously decrease UVA-induced cell death. Dissection of the UVA-induced signalling pathways revealed that TC-22 could suppress the activation of JNK and caspase 3, but not of ERK and p38. Reduction of UVA-induced cleavage of caspase 3 and sub-G1 phase accumulation by pretreatment of TC-22 was also observed. In zebrafish, we showed that UVA irradiation could decrease the survival and hatching rate, suppress heart beats of embryos and enhance the pigmentation of larvae. Pretreatment of TC-22 could significantly reverse UVA-induced the suppression in hatching of eggs and heart beating of embryos and also lowered the UVA-induced pigmentation in zebrafish. Collectively, we demonstrate that TC-22, a GFP chromophore derivative, can ameliorate the UVA-induced stress responses in both epidermal keratinocytes and zebrafish, suggesting the potential use of TC-22 in photoprotection in the future.