Chemiluminescence of 3‐aminophthalic acid anion–hydrogen peroxide–cobalt (II)

The 3‐aminophthalic acid anion is a light emitter in luminol chemiluminescence. In the present study, the chemiluminescence of the 3‐aminophthalic acid anion itself in the presence of hydrogen peroxide–cobalt (II) was studied. The results indicated that 3‐aminophthalic acid anion is highly chemiluminescent in the typical hydrogen peroxide–cobalt (II) system. The peak wavelength of this chemiluminescence and the kinetic profile of the 3‐aminophthalic acid anion–hydrogen peroxide–cobalt (II) reaction showed similarity with that of luminol, but the chemiluminescence of 3‐aminophthalic acid anion had a much lower background signal. In addition, the chemiluminescence mechanism of 3‐aminophthalic acid anion–hydrogen peroxide–cobalt (II) was also discussed and speculated as the interaction between 3‐aminophthalic acid anion and singlet oxygen.     

Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.

Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 568, and Alexa 594 dyes are a new series of fluorescent dyes with emission/excitation spectra similar to those of AMCA, Lucifer Yellow, fluorescein, rhodamine 6G, tetramethylrhodamine or Cy3, lissamine rhodamine B, and Texas Red, respectively (the numbers in the Alexa names indicate the approximate excitation wavelength maximum in nm). All Alexa dyes and their conjugates are more fluorescent and more photostable than their commonly used spectral analogues listed above. In addition, Alexa dyes are insensitive to pH in the 4-10 range. We evaluated Alexa dyes compared with conventional dyes in applications using various conjugates, including those of goat anti-mouse IgG (GAM), streptavidin, wheat germ agglutinin (WGA), and concanavalin A (ConA). Conjugates of Alexa 546 are at least twofold more fluorescent than Cy3 conjugates. Proteins labeled with the Alexa 568 or Alexa 594 dyes are several-fold brighter than the same proteins labeled with lissamine rhodamine B or Texas Red dyes, respectively. Alexa dye derivatives of phalloidin stain F-actin with high specificity. Hydrazide forms of the Alexa dyes are very bright, formaldehyde-fixable polar tracers. Conjugates of the Alexa 430 (ex 430 nm/em 520 nm) and Alexa 532 (ex 530 nm/em 548 nm) fluorochromes are spectrally unique fluorescent probes, with relatively high quantum yields in their excitation and emission wavelength ranges.     

Light from maillard reaction: Photon counting,emission spectrum,photography and visual perception

Several authors have reported on high-sensitivity measurement of oxygen-dependent low-level chemiluminescence (CL) from Maillard reactions (MR), i.e. nonenzymatic amino-carbonyl reactions between reducing sugars and amino acids (also referred to as nonenzymatic browning). Here we report for the first time, that light from Maillard reactions can be seen by the human eye and also can be photographed. In parallel with visual perception and photography CL was monitored by means of a CL-detection programme of a liquid scintillation counter (LSC, single photon rate counting). CL emission spectrum was recorded by a monochromator-microchannel plate photomultiplier arrangement. CL intensity from reaction of 6-aminocaproic acid with D-ribose (200 mg each) in 5 mL H₂O at pH 11 at 95°C was high enough for visual perception after adaptation to absolute darkness. Reaction in dimethylsulphoxide (DMSO) exhibited strongly enhanced CL (10 mg each in 5 mL were sufficient for visual detection) and could be photographed (15 minutes' exposure, ASA 6400); all characteristics of Maillard specific CL (O₂-dependence, no CL from nonreducing sugars, inhibition by sulphur compounds) remained. Visual detection of CL and measurement by LSC were in full concordance. The CL emission spectrum showed two broad peaks at around 500 nm and 695 nm. Fluorescence emission of the brown reaction mixture matched the bluegreen part of the CL emission spectrum. Emission of visible light during Maillard reactions may partly originate from oxygen-dependent generation of excited states and energy transfer to simultaneously formed fluorescent products of the browning reaction.     

The red‐edge effects: 30 years of exploration

In 1970, three laboratories independently made a discovery that, for aromatic fluorophores embedded into different rigid and highly viscous media, the spectroscopic properties do not conform to classical rules. The fluorescence spectra can depend on excitation wavelength, and the excited‐state energy transfer, if present, fails at the ‘red’ excitation edge. These red‐edge effects were related to the existence of excited‐state distribution of fluorophores on their interaction energy with the environment and the slow rate of dielectric relaxation of this environment. In these conditions the site‐selection can be provided by variation of the energy of illuminating light quanta, and the behaviour of selected species can be followed as a function of time and other variables. These observations found extensive application in different areas of research: colloid and polymer science, molecular biophysics, photochemistry and photobiology. In particular, they led to the development of very productive methods of studying the dynamics of dielectric relaxations in protein and membranes, using the tryptophan emission and the emission of a variety of probes. These studies were extended to the time domain with the observation of new site‐selective effects in emission intensity and anisotropy decays. They stimulated the emergence and development of cryogenic energy‐selective and single‐molecular techniques that became valuable tools in their own right in chemistry and biophysics research. Site‐selection effects were discovered for electron‐transfer and proton‐transfer reactions if they depended on the dynamics of the environment. This review is focused on the progress in the field of red‐edge effects, their applications and prospects. Copyright © 2002 John Wiley & Sons, Ltd.     

Magnesium methoxide‐induced chemiluminescent decomposition of bicyclic dioxetanes bearing a 2′‐alkoxy‐2‐hydroxy‐1,1′‐binaphthyl‐7‐yl moiety

Bicyclic dioxetanes 2a–c bearing a 2′‐alkoxy‐2‐hydroxy‐1,1′‐binaphthyl‐7‐yl moiety were effectively synthesized and their base‐induced chemiluminescent decomposition was investigated by the use of alkaline metal (Na⁺ and K⁺) or Mg²⁺ alkoxide in MeOH. When 2a–c were treated with tetrabutylammonium fluoride (TBAF) in dimethyl sulfoxide (DMSO) as a reference system, they showed chemiluminescence as a flash of orange light (maximum wavelength λ_max^CL = 573–577 nm) with efficiency Φ^CL = 6–8 × 10^–2. On the other hand, for an alkaline metal (Na⁺ or K⁺) alkoxide/MeOH system, 2a–c decomposed slowly to emit a glow of chemiluminescence, the spectra of which were shifted slightly toward red from the TBAF/DMSO system, and Φ^CL (= 1.4–2.3 × 10^–3) was considerably decreased. In addition, Mg(OMe)₂ was found to play a characteristic role as a base for the chemiluminescent decomposition of 2a–c through coordination to the intermediary oxidoaryl‐substituted dioxetanes 13. Thus, Mg²⁺ increased Φ^CL to more than twice those with Na⁺ or K⁺, while it shifted λ_max^CL considerably toward blue (λ_max^CL = 550–566 nm). Copyright © 2012 John Wiley & Sons, Ltd.     

Dual‐color‐emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy

We isolated and characterized a green fluorescent protein (GFP) from the sea cactus Cavernularia obesa. This GFP exists as a dimer and has absorption maxima at 388 and 498 nm. Excitation at 388 nm leads to blue fluorescence (456 nm maximum) at pH 5 and below, and green fluorescence (507 nm maximum) at pH 7 and above, and the GFP is remarkably stable at pH 4. Excitation at 498 nm leads to green fluorescence (507 nm maximum) from pH 5 to pH 9. We introduced five amino acid substitutions so that this GFP formed monomers rather than dimers and then used this monomeric form to visualize intracellular pH change during the phagocytosis of living cells by use of fluorescence microscopy. The intracellular pH change is visualized by use of a simple long‐pass emission filter with single‐wavelength excitation, which is technically easier to use than dual‐emission fluorescent proteins that require dual‐wavelength excitation. Copyright © 2013 John Wiley & Sons, Ltd.     

Flow‐injection chemiluminescence method for the determination of chloramphenicol based on luminol–sodium periodate order‐transform second‐chemiluminescence reaction

A new chemiluminescence (CL) reaction was observed when chloramphenicol solution was injected into the mixture after the end of the reaction of alkaline luminol and sodium periodate or sodium periodate was injected into the reaction mixture of chloramphenicol and alkaline luminol. This reaction is described as an order‐transform second‐chemiluminescence (OTSCL) reaction. The OTSCL method combined with a flow‐injection technique was applied to the determination of chloramphenicol. The optimum conditions for the order‐transform second‐chemiluminescence emission were investigated. A mechanism for OTSCL has been proposed on the basis of the chemiluminescence kinetic characteristics, the UV‐visible spectra and the chemiluminescent spectra. Under optimal experimental conditions, the CL response is proportional to the concentration of chloramphenicol over the range 5.0 × 10^?7–5.0 × 10^?5 mol/L with a correlation coefficient of 0.9969 and a detection limit of 6.0 × 10^?8 mol/L (3σ). The relative standard deviation (RSD) for 11 repeated determinations of 5.0 × 10^?6 mol/L chloramphenicol is 1.7%. The method has been applied to the determination of chloramphenicol in pharmaceutical samples with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.     

Chemiluminescence intensities and spectra of luminol oxidation by sodium hypochlorite in the presence of hydrogen peroxide

Hydrogen peroxide amplifies the chemiluminescence in the oxidation of luminol by sodium hypochlorite. A linear relationship between concentration of hydrogen peroxide and light intensity was found in the concentration range 5 × 10^?8?7.5 × 10^?6 mol/l. At 7.5 × 10^?6 mol/l H₂O₂ the chemiluminescence is amplified 550—fold. The chemiluminescence spectra of these reactions have a wavelength maximum at 431 nm independent of the concentration of hydrogen peroxide. The results indicate that hydrogen peroxide is a necessary component in the chemiluminescent oxidation of the luminol by sodium hypochlorite.     

Laser tissue welding analyzed using fluorescence,Stokes shift spectroscopy,and Huang‐Rhys parameter

Near infrared (NIR) continuous wave laser radiation at the 1,450 nm wavelength was used to weld porcine aorta and skin samples via the absorption of combitional vibrational modes of native water in the tissues. The fluorescence spectra were measured from the key native molecules of welded and non‐welded tissues at specific excitation and emission wavelengths from collagen, elastin, and tryptophan. The changes in the fluorescence intensities and differences in Stokes shift (Δν_ss) of key native fluorophores were measured to differentiate the Huang‐Rhys parameter values (S) of the chromophores. The strength of coupling depends on the local electron‐vibration intra‐tissue molecular environment and the amount of polar solvent water surrounding the net charges on collagen, elastin, and tryptophan. The S values for both non‐welded and welded tissues were almost the same and less than 3, suggesting minimal changes in the local molecular environment as a result of welding. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Unique interactions between the chromophore and glutamate 16 lead to far‐red emission in a red fluorescent protein

mPlum is a far‐red fluorescent protein with emission maximum at ～650 nm and was derived by directed evolution from DsRed. Two residues near the chromophore, Glu16 and Ile65, were previously revealed to be indispensable for the far‐red emission. Ultrafast time‐resolved fluorescence emission studies revealed a time dependent shift in the emission maximum, initially about 625 nm, to about 650 nm over a period of 500 ps. This observation was attributed to rapid reorganization of the residues solvating the chromophore within mPlum. Here, the crystal structure of mPlum is described and compared with those of two blue shifted mutants mPlum‐E16Q and ‐I65L. The results suggest that both the identity and precise orientation of residue 16, which forms a unique hydrogen bond with the chromophore, are required for far‐red emission. Both the far‐red emission and the time dependent shift in emission maximum are proposed to result from the interaction between the chromophore and Glu16. Our findings suggest that significant red shifts might be achieved in other fluorescent proteins using the strategy that led to the discovery of mPlum.     

Eu3+‐activated Y2MoO6: a narrow band red‐emitting phosphor with strong near‐UV absorption

Near‐UV excited narrow line red‐emitting phosphors, Eu³⁺‐activated Y₂MoO₆ systems, were synthesized using a simple molten salt reaction. The structure and photoluminescence characteristics were investigated using X‐ray powder diffraction, UV–Vis absorption and fluorescent spectrophotometry. The excitation spectra show strong broad‐band absorptions in the near‐UV to blue light regions which match the radiation of near‐UV light‐emitting diode chips well. Under excitation of either near‐UV or blue light, intense red emission with a main peak of 611 nm is observed, ascribed to the ⁵D₀–⁷F₂ transition of Eu³⁺ ions; the optimal doping concentration is 20 mol%. The chromaticity coordinates (x = 0.65, y = 0.34) of the as‐obtained phosphor are very close to the National Television Standard Committee standard values (x = 0.67, y = 0.33). All these characteristics suggest that this material is a promising red‐emitting phosphor candidate for white‐LEDs based on near‐UV LED chips. Copyright © 2012 John Wiley & Sons, Ltd.     

One‐step synthesis of cationic gold nanoclusters with high catalytic activity on luminol chemiluminescence reaction

The present study reports a one‐step synthesis method for the preparation of cationic gold nanoclusters (Au NCs). Polyethyleneimine (PEI), a positively charged hyperbranched polyamine, was selected as the capping reagent. Glutathione showed a synergistic effect on the formation of the small size of cationic Au NCs. The prepared cationic Au NCs have a size less than 2 nm and carry a positive charge in solution with pH less than 11. The cationic PEI–Au NCs‐triggered luminol chemiluminescence (CL) reactions showed slow and intense CL profiles. The maximum CL intensity can be obtained within 10 min and the CL signal maintained almost the same within 30 min. A linear increase of CL intensity was observed in the presence of an increasing concentration of cationic Au NCs ranging from 0.030 μM to 15 μM. The linear response of the cationic Au NCs in the CL reaction and the glow‐type CL profile make the proposed CL reaction have broad application prospects in the field of biological analysis and CL imaging.     

Vector Alkaline Phosphatase Substrate Blue III: One Substrate for Brightfield Histochemistry and High-resolution Fluorescence Imaging by Confocal Laser Scanning Microscopy

A number of histochemical chromogenic substrates for alkaline phosphatase are commercially available and give reaction products with a range of colours for brightfield examination. Some of these reaction products are also fluorescent, exhibiting a wide excitation range and a broad emission peak. We report here that one of these substrates, Vector Blue III, yields a stable, strongly fluorescent reaction product with an excitation peak around 500 nm and a large Stokes shift to an emission peak at 680 nm. The reaction product can be excited using a mercury lamp with a fluorescein excitation filter or an argon ion laser at 488 nm or 568 nm, and the emission detected using a long-pass filter designed for Cy-5. Thus, a single substrate is suitable for brightfield imaging of tissue sections and high-resolution analysis of subcellular detail, using a confocal laser scanning microscope, in the same specimen.     

Ultrafast liquid chromatographic analysis of erdosteine in human plasma based on fluorimetric detection and precolumn derivatization with 4‐bromomethyl‐7‐methoxycoumarin: Application to pharmacokinetic studies

In this study, a new analytical method for erdosteine (ERD) in plasma based on high‐performance liquid chromatography and a fluorimetric detector, is presented. Precolumn derivatization of ERD with 4‐bromomethyl‐7‐methoxy coumarin (BrMmC) and dibenzo‐18‐crown‐6‐ether as a reaction catalyst led to the production of a fluorescent compound. ERD was monitored by fluorescence with an excitation wavelength λ_ext. = 325 nm and emission wavelength λ_em. = 390 nm. Optimum reaction conditions were carefully studied and optimized. A chromatographic procedure was performed using a C18 column of 150 × 4.6 mm and 3 μm particle size and a mobile phase consisting of methanol:acetonitrile:water (30:30:40, v/v/v) under a flow rate of 0.5 ml min^?1. A calibration plot was established covering analyte concentration range 0.2–3.0 μg ml^?1; the detection limit was 0.015 μg ml^?1 and quantification limit was 0.05 μg ml^?1. Mean recovery was 87.33% and relative standard deviation was calculated to be less than 4.4%. The developed method was successfully used to determine pharmacokinetic preparations of ERD subsequent to administration of a 900 mg dose capsule to a healthy 40‐year‐old woman volunteer.     

Three‐dimensional cellular imaging in thick biological tissue with confocal detection of one‐photon fluorescence in the near‐infrared II window

Fluorescence imaging in the second near‐infrared optical window (NIR‐II, 900‐1700 nm) has become a technique of choice for noninvasive in vivo imaging in recent years. Greater penetration depths with high spatial resolution and low background can be achieved with this NIR‐II window, owing to low autofluorescence within this optical range and reduced scattering of long wavelength photons. Here, we present a novel design of confocal laser scanning microscope tailored for imaging in the NIR‐II window. We showcase the outstanding penetration depth of our confocal setup with a series of imaging experiments. HeLa cells labeled with PbS quantum dots with a peak emission wavelength of 1276 nm can be visualized through a 3.5‐mm‐thick layer of scattering medium, which is a 0.8% Lipofundin solution. A commercially available organic dye IR‐1061 (emission peak at 1132 nm), in its native form, is used for the first time, as a NIR‐II fluorescence label in cellular imaging. Our confocal setup is capable of capturing optically sectioned images of IR‐1061 labeled chondrocytes in fixed animal cartilage at a depth up to 800 μm, with a superb spatial resolution of around 2 μm.      

Caspase-3 sensitive signaling in vivo in apoptotic HeLa cells by chemically engineered intramolecular fluorescence resonance energy transfer mutants of green fluorescent protein

Green fluorescent protein (UV5) was re-engineered to remove native cysteine residues, and a new cysteine was introduced near the C-terminus, approximately 20 A from the native fluorophore, for site-specific attachment of chemical fluorophores. The resultant efficient intramolecular FRET quenched GFP emission and gave a new emission band from the conjugated fluorophore. Caspase-3 cleavage of constructs with a caspase-3 sequence near the C-terminus in the sequence between the native fluorophore and the new cysteine, located C-terminal to the caspase site, destroyed the FRET, the emitted color reverting to that of unmodified GFP. This process was demonstrated in vitro with caspase-3 and lysates from cells undergoing apoptosis. Real-time emission changes for the Alexa Fluor 532 conjugate of this GFP, studied quantitatively in vivo for single HeLa cells using the ratios of fluorescence at the red and green maxima by confocal microscopy, showed that caspase-3 action in the cytosol preceded that in the nucleus.     

A lab‐on‐a‐chip device for analysis of amlodipine in biological fluids using peroxyoxalate chemiluminescence system

A highly sensitive, rapid and economical method for the determination of amlodipine (AM) in biological fluids was developed using a peroxyoxalate chemiluminescence (CL) system in a lab‐on‐a‐chip device. Peroxyoxalate‐CL is an indirect type of CL that allows the detection of native fluorophores or compounds derivatized with fluorescent labels. Here, fluorescamine was reacted with AM, and the derivatization product was used in a bis‐(2,4,6‐trichlorophenyl)oxalate‐CL system. Fluorescamine reacts selectively with aliphatic primary amine at neutral or basic pH. As most of the calcium channel blocker and many cardiovascular drugs do not contain primary amine, the developed method is highly selective. The parameters that influenced the CL signal intensity were studied carefully. These included the chip geometry, pH, concentration of reagents used and flow rates. Moreover, we confirmed our previous observation about the effects of imidazole, which is commonly used in the bis‐(2,4,6‐trichlorophenyl)oxalate‐CL system as a catalyst, and found that the signal was significantly improved when imidazole was absent. Under optimized conditions, a calibration curve was obtained with a linear range (10–100 µg/L). The limit of detection was 3 µg/L, while the limit of quantification was 10 µg/L. Finally the method was applied for the determination of AM in biological fluids successfully. Copyright © 2014 John Wiley & Sons, Ltd.     

Blue excitable green emitting Ce3+ doped CaS phosphor for w‐LEDs

CaS:Ce³⁺ is an efficient green‐emitting (535 nm) phosphor, excitable with blue light (450–470 nm) and was synthesized via a solid‐state reaction method by heating under a reducing atmosphere. The luminescent properties, photoluminescent (PL) excitation and emission of the phosphor were analyzed by spectrofluorophotometry. The excitation and emission peaks of the CaS:Ce³⁺ phosphor lay in the visible region, which made them relevant for light‐emitting diode (LED) application for the generation of white light. Judd‐Oflet parameters were calculated and revealed that green light emitted upon blue illumination. The prepared phosphor had strong blue absorption at 470 nm and a broad green emission band range from 490–590 nm with the peak at 537 nm. The characteristics of the CaS:Ce³⁺ phosphor make it suitable for use as a wavelength tunable green emitting phosphor for three band white LEDs pumped by a blue LED (470 nm). The Commission International de l'Eclairage co‐ordinates were calculated by a spectrophotometric method using the spectral energy distribution (0.304, 0.526) and confirm the green emission. The potential application of this phosphor is as a phosphor‐converted white light‐emitting diode. Copyright © 2015 John Wiley & Sons, Ltd.     

Preparation and application of solvent‐modulated self‐doped N–S multicolour fluorescence carbon quantum dots

In this paper, two types of carbon quantum dot (CQDs) were prepared using biocompatible l ‐methionine as the carbon source and urea as the nitrogen source and a one‐step hydrothermal treatment. By changing the reaction solvents (deionized (DI) water and dimethylformamide (DMF)), the maximum emission of the resulting CQDs shifted from blue to red light. Specifically, the emission wavelength of the CQDs moved from 433 nm to 625 nm following embedding of a new functional group (–CONH–) on the surface of the CQDs. Photoluminescence quantum yields of the CQDs with blue and red emission reached 64% and 61%, respectively. The R‐CQDs were used to detect metal ions and a linear relationship was demonstrated between ln(F/F₀) and Fe³⁺ concentration in the range 0–0.5 mmol/L with a detection limit of 0.067 μM. Therefore these R‐CQDs have great potential as fluorescent probes for Fe³⁺ detection. We expect that the excellent water‐soluble, biocompatible and optical properties of the CQDs developed in this work mean that they will be widely used to detect biological cells.     

Chemiluminescence of 4‐styrylphthal‐hydrazides with crown ether as ionophore

Two 4‐styrylphthalhydrazides bearing crown ether moieties as ionophores were prepared and their chemiluminescence (CL) behaviour was investigated. Their aerobic oxidation in the presence of excess tert‐BuOK in DMSO provided the CL with much shorter emission wavelengths than those of the corresponding phthalate dianions, indicating that the emission arose from the excited phthalates incorporated with potassium ions at the ionophore sites. Such incorporation of potassium ions with the crown sites also reduced the fluorescence intensities of the phthalate dianions which caused an energy transfer CL, resulting in the additional emissions of the phthalhydrazide monoanions in much longer wavelength regions. On the other hand, only the emission from the excited phthalate dianion was detected under aqueous conditions using aqueous hydrogen peroxide in acetonitrile as the oxidant, but no meaningful difference in the CL intensity depending on the kind of metal cations was observed. Copyright © 2008 John Wiley & Sons, Ltd.