Fluorescent Chromophore Research Articles

Dual-Emission GFP Chromophore-Based Derivative for Imaging and Discriminating Aβ Oligomers and Aggregates

β-Amyloid deposition is one of the main pathological features of Alzheimer's disease (AD). The development of fluorescent probes targeting specific β-amyloid species has recently become an attractive strategy to achieve the early diagnosis of AD. In this work, a dual-channel fluorescent protein chromophore derivative C17 was rationally designed and synthesized for the detection and discrimination of Aβ42 aggregates and oligomers. C17 exhibits a specific turn-on emission peak for Aβ42 oligomers at ∼470 nm (peak A) and a peak at ∼600 nm (peak B) for both Aβ42 oligomers and Aβ42 aggregates. Taking advantage of the dual emission of the probe, the dynamic aggregation process of the Aβ42 peptide was monitored in solution. Moreover, double staining of brain sections from transgenic AD mice revealed that peak A of C17 preferentially detected Aβ42 oligomers, whereas peak B was more sensitive to Aβ42 aggregates. The fact that probe C17 can be used for dissecting these two Aβ42 species makes C17 a comprehensive tool for β-amyloid aggregation studies in AD research.

Post translational modifications of Trifolitoxin: a blue fluorescent peptide antibiotic

Trifolitoxin (TFX, C41H63N15O15S) is a selective, ribosomally-synthesized, post-translationally modified, peptide antibiotic, produced by Rhizobium leguminosarum bv. trifolii T24. TFX specifically inhibits α-proteobacteria, including the plant symbiont Rhizobium spp., the plant pathogen Agrobacterium spp. and the animal pathogen Brucella abortus. TFX-producing strains prevent legume root nodulation by TFX-sensitive rhizobia. TFX has been isolated as a pair of geometric isomers, TFX1 and TFX2, which are derived from the biologically inactive primary amino acid sequence: Asp-Ile-Gly-Gly-Ser-Arg-Gln-Gly-Cys-Val-Ala. Gly-Cys is present as a thiazoline ring and the Arg-Gln-Gly sequence is extensively modified to a UV absorbing, blue fluorescent chromophore. The chromophore consists of a conjugated, 5-membered heterocyclic ring and side chain of modified glutamine.

A novel violet fluorescent protein contains a unique oxidized tyrosine as the simplest chromophore ever reported in fluorescent proteins.

We describe an engineered violet fluorescent protein from the lancelet Branchiostoma floridae (bfVFP). This is the first example of a GFP-like fluorescent protein with a stable fluorescent chromophore lacking an imidazolinone ring; instead, it consists of oxidized tyrosine 68 flanked by glycine 67 and alanine 69. bfVFP contains the simplest chromophore reported in fluorescent proteins and was generated from the yellow protein lanFP10A2 by two synergetic mutations, S148H and C166I. The chromophore structure was confirmed crystallographically and by high-resolution mass spectrometry. The photophysical characteristics of bfVFP (323/430 nm, quantum yield 0.33, and Ec 14,300 M-1 cm-1 ) make it potentially useful for multicolor experiments to expand the excitation range of available FP biomarkers and Förster resonance energy transfer with blue and cyan fluorescent protein acceptors.

Transforming polyethylene and polypropylene into nontraditional fluorescent polymers by thermal oxidation

Polyethylene and polypropylene, which do not contain any fluorescent chromophores, are transformed into nontraditional photoluminescent materials with strong fluorescence emissions by an extremely simple thermal oxidation method.

Environment-sensitive fluorogens based on a GFP chromophore structural motif

In this paper, we present a new series of arylidene-imidazolones with a structural motif of Green Fluorescent Protein chromophore with a pronounced solvent-dependent FQY variation. The identified structure-activity patterns made possible creation of a series of fluorogenic dyes with various positions of spectral maxima and Stokes shift. The developed dyes are cell-permeable and can be used for selective staining of the endoplasmic reticullum in living eukaryotic cells.

Nearly 100% energy transfer at the interface of metal-organic frameworks for X-ray imaging scintillators

<h2>Summary</h2> In this work, we describe a highly efficient and reabsorption-free X-ray-harvesting system using luminescent metal-organic framework (MOF)-fluorescence chromophore composite films. The ultrafast time-resolved experiments and density functional theory calculations demonstrate that a nearly 100% energy transfer from a luminescent MOF with a high atomic number to an organic chromophore with thermally activated delayed fluorescence (TADF) character can be achieved. Such an unprecedented efficiency of interfacial energy transfer and the direct harnessing of singlet and triplet excitons of the TADF chromophore led to remarkable enhancement of radioluminescence upon X-ray radiation. A low detection limit of 256 nGy/s of the fabricated X-ray imaging scintillator was achieved, about 60 times lower than the MOF and 7 times lower than the organic chromophore counterparts. More importantly, this detection limit is about 22 times lower than the standard dosage for a medical examination, making it an excellent candidate for X-ray radiography.

Ultrafast Aggregation-Induced Tunable Emission Enhancement in a Benzothiadiazole-Based Fluorescent Metal-Organic Framework Linker.

Aggregation-induced emission enhancement (AIEE) is a process recently exploited in solid-state materials and organic luminophores, and it is explained by tight-molecular packaging. However, solution-phase AIEE and its formation mechanism have not been widely explored. This work investigated AIEE phenomena in two donor-acceptor-donor-type benzodiazole-based molecules (the organic building block in metal-organic frameworks) with an acetylene and phenyl π-conjugated backbone tapered with a carboxylic acid group at either end. This was done using time-resolved electronic and vibrational spectroscopy in conjunction with time-dependent density functional theory (TD-DFT) calculations. Fluorescence up-conversion spectroscopy and time-correlated single-photon counting conclusively showed an intramolecular charge transfer-driven aggregate emission enhancement. This is shown by a red spectral shift of the emission spectra as well as an increase in the fluorescence lifetime from 746 ps at 1.0 × 10-11 to 2.48 ns at 2.0 × 10-3 M. The TD-DFT calculations showed that a restricted intramolecular rotation mechanism is responsible for the enhanced emission. The femtosecond infrared (IR) transient absorption results directly revealed the structural dynamics of aggregate formation, as evident from the evolution of the C≡C vibrational marker mode of the acetylene unit upon photoexcitation. Moreover, the IR data clearly indicated that the aggregation process occurred over a time scale of 10 ps, which is consistent with the fluorescence up-conversion results. Interestingly, time-resolved results and DFT calculations clearly demonstrated that both acetylene bonds and the sulfur atom are the key requirements to achieve such a controllable aggregation-induced fluorescence enhancement. The finding of the work not only shows how slight changes in the chemical structure of fluorescent chromophores could make a tremendous change in their optical behavior but also prompts a surge of research into a profound understanding of the mechanistic origins of this phenomenon. This may lead to the discovery of new chemical strategies that aim to synthesize novel chromophores with excellent optical properties for light-harvesting applications.

Detecting the insoluble protein aggregates in live cells using an AIE derivative of fluorescent protein chromophore

Many incurable or unmanageable human protein conformational diseases are associated with the misfolding or aggregation of the aberrantly processed or mutant proteins. In this work, we report an aggregation-induced emission (AIE) derivative of fluorescent protein chromophore used to detect the insoluble protein aggregates in live cells. Based on the 4-hydroxybenzylidene-imidazolinone (HBI), we designed and synthesized a series of AIEgens that span a wide range of viscosity coefficients (χ), thus mimicking the viscous microenvironment of a wide variety of amorphous protein aggregates. The mechanism of these AIEgens were systematically investigated using a combination of photophysical studies, computational analyses and structure-function studies. With the aid of the AggTag method, the optimized probe was used to realize the imaging of the aggregated proteome under the control of the proteostasis network. Besides, we also described the formation and decomposition of protein aggregates under the control of small molecule proteostasis regulators. Briefly, we developed a series of AIEgens that explore varying viscosity sensitivities to visualize protein aggregation in live cells as well as study other biological processes that associated with local viscosity changes.

Pyrene-Benzimidazole Derivatives as Novel Blue Emitters for OLEDs.

Three novel small organic heterocyclic compounds: 2-(1,2-diphenyl)-1H-benzimidazole-7-tert-butylpyrene (compound A), 1,3-di(1,2-diphenyl)-1H-benzimidazole-7-tert-butylpyrene (compound B), and 1,3,6,8-tetra(1,2-diphenyl)-1H-benzimidazolepyrene (compound C) were synthesized and characterized for possible applications as blue OLED emitters. The specific molecular design targeted decreasing intermolecular aggregation and disrupting crystallinity in the solid-state, in order to reduce dye aggregation, and thus obtain efficient pure blue photo- and electroluminescence. Accordingly, the new compounds displayed reasonably high spectral purity in both solution- and solid-states with average CIE coordinates of (0.160 ± 0.005, 0.029 ± 0.009) in solution and (0.152 ± 0.007, 0.126 ± 0.005) in solid-state. These compounds showed a systematic decrease in degree of crystallinity and intermolecular aggregation due to increasing steric hindrance, as revealed using powder X-ray diffraction analysis and spectroscopic studies. An organic light-emitting diode (OLED) prototype fabricated using compound B as the non-doped emissive layer displayed an external quantum efficiency (EQE) of 0.35 (±0.04)% and luminance 100 (±6) cd m−2 at 5.5 V with an essentially pure blue electroluminescence corresponding to CIE coordinates of (0.1482, 0.1300). The highest EQE observed from this OLED prototype was 4.3 (±0.3)% at 3.5 V, and the highest luminance of 290 (±10) cd m−2 at 7.5 V. These values were found comparable to characteristics of the best pure blue OLED devices based on simple fluorescent small-molecule organic chromophores.

Development of phenothiazine-based fluorescent probe with aggregation induced emission (AIE) for detection of hydrazine and its application in imaging of living cells

A new fluorescent probe CMPA based on phenothiazine for detection of hydrazine was designed and synthesized. The probe employed methyl cyanoacetate as the recognition group and phenothiazine as the fluorescent chromophore. The Stoke shift and the detection limit of the probe CMPA at the presence of hydrazine reached to 100 nm and 5.2 ppb respectively. In addition, the probe CMPA dyed the HepG2 cells with violet colour at the presence of hydrazine.

Action spectroscopy of the isolated red Kaede fluorescent protein chromophore.

Incorporation of fluorescent proteins into biochemical systems has revolutionized the field of bioimaging. In a bottom-up approach, understanding the photophysics of fluorescent proteins requires detailed investigations of the light-absorbing chromophore, which can be achieved by studying the chromophore in isolation. This paper reports a photodissociation action spectroscopy study on the deprotonated anion of the red Kaede fluorescent protein chromophore, demonstrating that at least three isomers-assigned to deprotomers-are generated in the gas phase. Deprotomer-selected action spectra are recorded over the S1 ← S0 band using an instrument with differential mobility spectrometry coupled with photodissociation spectroscopy. The spectrum for the principal phenoxide deprotomer spans the 480-660nm range with a maximum response at ≈610nm. The imidazolate deprotomer has a blue-shifted action spectrum with a maximum response at ≈545nm. The action spectra are consistent with excited state coupled-clustercalculations of excitation wavelengths for the deprotomers. A third gas-phase species with a distinct action spectrum is tentatively assigned to an imidazole tautomer of the principal phenoxide deprotomer. This study highlights the need for isomer-selective methods when studying the photophysics of biochromophores possessing several deprotonation sites.

Singlet oxygen-based photoelectrochemical detection of DNA

The current work, designed for the photoelectrochemical detection of DNA, evaluates light-responsive DNA probes carrying molecular photosensitizers generating singlet oxygen (1O2). We take advantage of their chromophore’s ability to produce 1O2 upon photoexcitation and subsequent photocurrent response. Type I, fluorescent and type II photosensitizers were studied using diode lasers at 406 nm blue, 532 nm green and 659 nm red lasers in the presensce and absence of a redox reporter, hydroquinone (HQ). Only type II photosensitizers (producing 1O2) resulted in a noticeable photocurrent in 1–4 nA range upon illumination, in particular, dissolved DNA probes labeled with chlorin e6 and erythrosine were found to give a well-detectable photocurrent response in the presence of HQ. Whereas, Type I photosensitizers and fluorescent chromophores generate negligible photocurrents (<0.15 nA). The analytical performance of the sensing system was evaluated using a magnetic beads-based DNA assay on disposable electrode platforms, with a focus to enhance the sensitivity and robustness of the technique in detecting complementary DNA targets. Amplified photocurrent responses in the range of 70–100 nA were obtained and detection limits of 17 pM and 10 pM were achieved using magnetic beads-captured chlorin e6 and erythrosine labeled DNA probes respectively. The presented novel photoelectrochemical detection can further be optimized and employed in applications for which enzymatic amplification such as polymerase chain reaction (PCR) is not applicable owing to their limitations and as an effective alternative to colorimetric detection when rapid detection of specific nucleic acid targets is required.

Modulation of fluorescent protein chromophore for photodynamic therapy and two-photon fluorescent imaging in living cells

The green fluorescent protein (GFP) chromophore has been widely applied as a biological marker or viscosity sensor. However, modulation of GFP chromophore for photodynamic therapy (PDT) has rarely been reported. Inspired by this, through the unique phenothiazine-anchored strategy, a near-infrared emission photosensitizer Lys-PtzFP was designed and synthesized to achieve this process. The maximum absorption wavelength of Lys-PtzFP was 460 nm and the corresponding molar extinction coefficient was 6.60 × 104 M−1 cm−1 in MeOH. The maximum emission wavelengths of Lys-PtzFP was at 725 nm. The photosensitizer Lys-PtzFP showed a high singlet oxygen quantum yield (ΦΔ = 0.42) and low fluorescence quantum yield (φF = 0.002) in MeOH. Besides, Lys-PtzFP possessed high specificity for lysosomes with negligible dark cytotoxicity (MTT assay >94.6%), good photostability and low half-maximal inhibitory concentration (IC50) of 0.93 μM. Finally, the intracellular photodynamic therapy experiments revealed that Lys-PtzFP can produce singlet oxygen in lysosomes and induce apoptosis under irradiation. In conclusion, Lys-PtzFP could be a promising photosensitizer for PDT and two-photon imaging applications. This type of photosensitizer can inspire the development of new photosensitizers for further explore highly efficient PDT.

Isopyrazole‐Masked Tetraketone: Tautomerism and Functionalization for Fluorescent Metal Ligands

AbstractAn isopyrazole (4H‐pyrazole)‐embedded nonane‐2,4,6,8‐tetraone derivative was synthesized as a masked‐tetraketone for stepwise and multiple functionalization. The masked‐tetraketone has six possible tautomers, but exists predominantly as a 12π‐electron conjugated keto‐enamine‐imine‐enol form, which was also observed in the solid state by single crystal X‐ray analysis. Stepwise functionalization to introduce metal‐coordination and fluorescence‐enhancement sites into the masked tetraketone furnished a fluorescent chromophore that indicates metal coordination on a microgram scale by considerably changing the solid‐state emission.

A new dual-functional chemsensor for the trace detection of mercury ion and imaging of hypochloric acid

Herein, a new dual-functional chemsensor 2-amino-3-(((E)-(5-(9-phenyl-9H-carbazol-3-yl)thiophen-2-yl)methylene)amino)maleonitrile (abbreviated as FDW) was designed and prepared for the trace detection mercury ion and imaging of hypochloric acid by two different fluorescence channels. The yellow-green channel of FDW was turned on within 6 min after adding trace-level Hg2+ under 365 nm UV lamp irradiation. For the first time, we proved that the chelation of mercury ions with FDW weakened the binding ability of CN, making it easy to break down under UV irradiation and generate the strong fluorescent chromophores. The detection limit was calculated to be as low as 0.34 nM, which demonstrated its potential applications in actual food samples. Interestingly, upon the addition of ClO−, the blue emission of FDW was significantly increased along with color change (from pale yellow to colorless). Under optimal conditions, the calibration curve is linear in the wide concentration range (0–240 μM), and the detection limit was 107 nM. In addition, FDW was successfully used for fluorescence imaging of ClO− in RAW 246.7 cells.

Frontispiece: BODIPYs with Photoactivatable Fluorescence

The integration of fluorescent BODIPY chromophores and photocleavable oxazine heterocycles within the same covalent skeleton results in the realization of molecules with photoswitchable fluorescence. The photochemical and photophysical properties of these compounds can be engineered to enable the monitoring of dynamics events in live embryos, the encoding of optical signals in live nematodes, the optical writing of fluorescent patterns and the imaging of live cells with sub-diffraction resolution. For more information, see the Minireview by Y. Zhang, F.M. Raymo et al. on page 11257

Designed Artificial Protein Heterodimers With Coupled Functions Constructed Using Bio-Orthogonal Chemistry.

The formation of protein complexes is central to biology, with oligomeric proteins more prevalent than monomers. The coupling of functionally and even structurally distinct protein units can lead to new functional properties not accessible by monomeric proteins alone. While such complexes are driven by evolutionally needs in biology, the ability to link normally functionally and structurally disparate proteins can lead to new emergent properties for use in synthetic biology and the nanosciences. Here we demonstrate how two disparate proteins, the haem binding helical bundle protein cytochrome b 562 and the β-barrel green fluorescent protein can be combined to form a heterodimer linked together by an unnatural triazole linkage. The complex was designed using computational docking approaches to predict compatible interfaces between the two proteins. Models of the complexes where then used to engineer residue coupling sites in each protein to link them together. Genetic code expansion was used to incorporate azide chemistry in cytochrome b 562 and alkyne chemistry in GFP so that a permanent triazole covalent linkage can be made between the two proteins. Two linkage sites with respect to GFP were sampled. Spectral analysis of the new heterodimer revealed that haem binding and fluorescent protein chromophore properties were retained. Functional coupling was confirmed through changes in GFP absorbance and fluorescence, with linkage site determining the extent of communication between the two proteins. We have thus shown here that is possible to design and build heterodimeric proteins that couple structurally and functionally disparate proteins to form a new complex with new functional properties.

A Family of Highly Fluorescent and Membrane-Permeable Bis(BF2) Acyl-Pyridinylhydrazine Dyes with Strong Solid-State Emission and Large Stokes Shifts: The BOAPH Fluorophores.

Organic small-molecule fluorescent chromophores have become essential to modern chemical, biological, and materials related investigations. Herein, a straightforward synthesis and subsequent borylation were presented to form a novel family of bisBF2-anchoring acyl-pyridinylhydrazine, which we named BOAPH. The chromophore enjoys outstanding structural diversities owing to varied acyl chlorides and N-heteroarenylhydrazides. These resultant BOAPH dyes are confirmed by NMR, HRMS, and single-crystal X-ray structure analysis. Their spectroscopic properties were studied, and most of the strong absorbance and bright fluorescence with maximum wavelengths centered in the range of 400 and 650 nm. More importantly, they exhibit promising fluorescence quantum yields up to 0.79 in solution and solid states, good photostability, and large Stokes shifts. Furthermore, a respective BOAPH dye with a para-dimethylaminophenyl group exhibited the interesting ability of ultrafast staining and two-photon imaging, which can specifically label lipid droplets of living cells immediately without the need for incubation.

Dramatically Enhanced and Red-shifted Photoluminescence Achieved by Introducing an Electron-withdrawing Group into a Non-traditional Luminescent Small Organic Compound.

Small organic compounds without any traditional fluorescent chromophores are generally non-emissive, and only very few are reported to emit weak blue fluorescence. Here we synthesized a non-traditional luminescent small organic compound N-(2,2,2-trifluoroethyl)acrylamide (TFAM) with dramatically enhanced and red-shifted photoluminescence by introducing a strong electron-withdrawing group into acrylamide (AM). Very impressively, TFAM emits cyan (472 nm) and yellow-green (560 nm) fluorescence in solutions and solid state, respectively. TFAM also shows aggregation-induced emission enhancement (AIEE) and excitation-dependent fluorescence (EDF) characteristics, as well as temperature and metal cations-responsive fluorescence. Theoretical calculations show that the introduction of electron-withdrawing group leads to a lower energy gap between the HOMO-LUMO energy levels in TFAM than in AM. And strong cooperative hydrogen bonds are formed in TFAM molecules, resulting in rigidification of molecular conformations. The study provides a strategy for preparing non-traditional luminescent compounds with enhanced and red-shifted photoluminescence.

Measurement report: Long-emission-wavelength chromophores dominate the light absorption of brown carbon in aerosols over Bangkok: impact from biomass burning

Abstract. Chromophores represent an important portion of light-absorbing species, i.e., brown carbon. Yet knowledge of what and how chromophores contribute to aerosol light absorption is still sparse. To address this problem, we examined soluble independent chromophores in a set of year-round aerosol samples from Bangkok. The water-soluble fluorescent chromophores identified via excitation–emission matrix (EEM) spectroscopy and follow-up parallel factor analysis could be mainly assigned as humic-like substances and protein-like substances, which differed in their EEM pattern from that of the methanol-soluble fraction. The emission wavelength of fluorescent chromophores in environmental samples tended to increase compared with that of the primary combustion emission, which could be attributed to secondary formation or the aging process. Fluorescent indices inferred that these light-absorbing chromophores were not significantly humified and comprised a mixture of organic matter of terrestrial and microbial origin, which exhibited a different characteristic from primary biomass burning and coal-combustion results. A multiple linear regression analysis revealed that larger fluorescent chromophores that were oxygen-rich and highly aromatic with high molecular weights were the key contributors of light absorption, preferably at longer emission wavelengths (λmax⁡ &gt; 500 nm). Positive matrix factorization analysis further suggested that up to 50 % of these responsible chromophores originated from biomass burning emissions.