Molecular Fluorophores Research Articles

AggFluor: Fluorogenic Toolbox Enables Direct Visualization of the Multi-Step Protein Aggregation Process in Live Cells.

Aberrantly processed or mutant proteins misfold and assemble into a variety of soluble oligomers and insoluble aggregates, a process that is associated with an increasing number of diseases that are not curable or manageable. Herein, we present a chemical toolbox, AggFluor, that allows for live cell imaging and differentiation of complex aggregated conformations in live cells. Based on the chromophore core of green fluorescent proteins, AggFluor is comprised of a series of molecular rotor fluorophores that span a wide range of viscosity sensitivity. As a result, these compounds exhibit differential turn-on fluorescence when incorporated in either soluble oligomers or insoluble aggregates. This feature allows us to develop, for the first time, a dual-color imaging strategy to distinguish unfolded protein oligomers from insoluble aggregates in live cells. Furthermore, we have demonstrated how small molecule proteostasis regulators can drive formation and disassembly of protein aggregates in both conformational states. In summary, AggFluor is the first set of rationally designed molecular rotor fluorophores that evenly cover a wide range of viscosity sensitivities. This set of fluorescent probes not only change the status quo of current imaging methods to visualize protein aggregation in live cells but also can be generally applied to study other biological processes that involve local viscosity changes with temporal and spatial resolutions.

Oil-Dispersible Green-Emitting Carbon Dots: New Insights on a Facile and Efficient Synthesis

Carbon dots (CDs) have been progressively attracting interest as novel environmentally friendly and cost-effective luminescent nanoparticles, for implementation in light-emitting devices, solar cells, photocatalytic devices and biosensors. Here, starting from a cost-effective bottom-up synthetic approach, based on a suitable amphiphilic molecule as carbon precursor, namely cetylpyridinium chloride (CPC), green-emitting CDs have been prepared at room temperature, upon treatment of CPC with concentrated NaOH solutions. The investigated method allows the obtaining, in one-pot, of both water-dispersible (W-CDs) and oil-dispersible green-emitting CDs (O-CDs). The study provides original insights into the chemical reactions involved in the process of the carbonization of CPC, proposing a reliable mechanism for the formation of the O-CDs in an aqueous system. The ability to discriminate the contribution of different species, including molecular fluorophores, allows one to properly single out the O-CDs emission. In addition, a mild heating of the reaction mixture, at 70 °C, has demonstrated the ability to dramatically decrease the very long reaction time (i.e. from tens of hours to days) at room temperature, allowing us to synthesize O-CDs in a few tens of minutes while preserving their morphological and optical properties.

Design, Synthesis, and Application of a Small Molecular NIR-II Fluorophore with Maximal Emission beyond 1200 nm

Small molecular fluorophores in the second near-infrared window (NIR-II) have aroused much interest because of their excellent performance. Herein, a new small molecular NIR-II fluorophore, FM1210, with maximal emission beyond 1200 nm is reported. Compared to the corresponding control fluorophore CF1065, FM1210 exhibits an increase of 145 nm in the emission maximum, which is ascribed to the simultaneous introduction of both a Se atom and amino groups into the benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) skeleton. This large increase in the maximal emission enables FM1210 to be capable of in vivo imaging with lower autofluorescence, higher signal-to-background ratio, and better resolution. Moreover, nanosized FM1210 encapsulated in liposomes possesses passive targeting ability and good water solubility, and is suitable for imaging a tumor and even its vasculature with high signal-to-background ratio.

Molecular Fluorophores Self-Organize into C-Dot Seeds and Incorporate into C-Dot Structures.

Various molecular fluorophores have been identified to be present during carbon-dot (C-dot) syntheses. However, the organization of such fluorophores in C-dots is still unknown. We study the self-assembly of 5-oxo-1,2,3,5-tetrahydroimidazo-[1,2-α]-pyridine-7-carboxylic acid (IPCA), a molecular fluorophore present during the synthesis of C-dots from citric acid and ethylenediamine. Both forms of IPCA (neutral and anionic) show a tendency to self-assemble into stacked systems, forming seeds of C-dots during their synthesis. IPCA also interacts with graphitic C-dot building blocks, fragments easily, and incorporates into their structures via π-π stacking. Both IPCA forms are able to create adlayers internally stabilized by an extensive hydrogen bonding network, with an arrangement of layers similar to that in ordinary graphitic C-dots. The results show the tendency of molecular fluorophores to form organized stacked seeds of C-dots and incorporate into C-dot structures. Such noncovalent structures can be further covalently interlinked via the carbonization process during C-dot growth.

Solvent Effect on Structural Elucidation of Photoluminescent Graphitic Carbon Nanodots.

Photoluminescence (PL) of carbon nanodots (CNDs) is proposed to originate from the polycyclic aromatic carbon-core and in situ synthesized molecular fluorophores. This work reports the CNDs prepared by direct pyrolysis of citric acid only at a prolonged time, 40 h, and their fluorescence emission parameters in a variety of solvents by steady-state and time-resolved emission spectroscopies. The response of fluorescence emission lifetime and emission quenching rate constants to changes in solvent parameters such as polarity and tumbling lifetime were essentially independent, unlike molecular fluorophores that display solvent-dependent emission parameters. Fluorescence emission was quenched in nitromethane additionally indicating to the polycyclic aromatic carbon-core as a predominant structural feature of the CNDs. The quenching of CND emission in the presence of benzophenone that has a strong triplet component in the excited state was observed. Quenching demonstrates the Stern–Volmer behavior and reveals the additional nonradiative decay pathways of CNDs. The main photophysical features of CNDs are discussed in terms of fluorescence emission originating from the excited state of the polycyclic aromatic carbon-core where contribution from the potential molecular fluorophores is considered minimal.

Molecular Fluorophores for Deep-Tissue Bioimaging.

Fluorescence imaging has made tremendous inroads toward understanding the complexity of biological systems, but in vivo deep-tissue imaging remains a great challenge due to the optical opacity of biological tissue. Recent improvements in laser and detector manufacturing have allowed the expansion of nonlinear and linear fluorescence imaging to the underexplored “tissue-transparent” second near-infrared (NIR-II; 1000–1700 nm) window, opening up new opportunities for optical access deep inside opaque tissue. Molecular fluorophores have historically played a major role in fluorescence bioimaging. It is increasingly important to design new molecular fluorophores to fully unlock the potential of NIR-II imaging techniques. In this outlook, we give an overview of the novel molecular fluorophores developed for deep-tissue bioimaging in the past five years and discuss their pros and cons in applications. Guidelines for designing new molecular fluorophores with the desirable properties are also provided.

High brightness NIR-II nanofluorophores based on fused-ring acceptor molecules

It is challenging to develop molecular fluorophores in the second near-infrared (NIR-II) window with long wavelength emission and high brightness, which can improve the performance of biological imaging. Herein, we report a molecular engineering approach to afford NIR-II fluorophores with these merits based on fused-ring acceptor (FRA) molecules. Dioctyl 3,4-propylenedioxy thiophene (PDOT-C8) is utilized as the bridging donor to replace 3-ethylhexyloxy thiophene (3-EHOT), leading to more than 20 times enhancement of brightness. The nanofluorophores (NFs) based on the optimized CPTIC-4F molecule exhibit an emission peak of 1,110 nm with a fluorescence quantum yield (QY) of 0.39% (QY of IR-26 is 0.050% in dichloroethane as reference) and peak absorption coefficient of 14.5 × 104 M−1cm−1 in aqueous solutions, which are significantly higher than those of 3-EHOT based COTIC-4F NFs. It is found that PDOT-C8 can weaken intermolecular aggregation, enhance protection of molecular backbone from water, and decrease backbone distortion, beneficial for the high brightness. Compared with indocyanine green with same injection dose, CPTIC-4F NFs show 10 times higher signal-to-background ratio for whole body vessels imaging at 1,300 nm long pass filters.

Conformational Behavior and Optical Properties of a Fluorophore Dimer as a Model of Luminescent Centers in Carbon Dots

The explanation of the origin of the fluorescence properties of carbon dots (CDs) represents an important task because of the great interest in the promising capabilities of these nanomaterials. 5-Oxo-1,2,3,5-tetrahydroimidazo-[1,2-α]-pyridine-7-carboxylic acid (IPCA), a molecular fluorophore, which is being created during the synthesis of CDs from citric acid and ethylenediamine, has been identified as an origin for the fluorescence of CDs. Using a combination of computational methods, we analyzed the UV absorption and fluorescence properties of the IPCA monomer and stacked IPCA dimers as basic models for the fluorescent centers in CDs. Density functional theory (DFT) for the ground state and time-dependent DFT calculations for excited states have been performed for the gas phase and for aqueous solution using a polarized continuum model. Classical molecular dynamics (MD) simulations of the dimer in the ground state have been carried out as well to investigate spontaneous association processes of IPCA and to analyze the ground state dynamics. Due to the complex charge distribution of the monomer and various possibilities of forming hydrogen bonds, in total, seven dimer structures have been identified at the DFT level as ground state minima with similar energies. Stabilities have been confirmed by domain-based local pair natural orbital (DLPNO) coupled cluster with singles and doubles and perturbative triples CCSD(T) calculations. The MD simulations confirm this picture, showing rotational flexibility processes of the two monomers with respect to each other. The lowest excited states have been characterized in terms of their orbital excitations and excitonic splitting. These calculations demonstrate the dominance of monomer π → π* transitions for the explanation of the observed UV spectra. Optimization of the dimer in the excited state did not lead to well-defined single structures but shows the picture of either intersection pathways to S1/S0 crossings which would quench fluorescence or stacked dimers with red-shifted fluorescence in comparison to the UV absorption. In the gas phase, both types of processes have been observed, whereas in solution only the stacked structures were found without any non-adiabatic radiationless deactivation processes.

Green Synthesis of Superior Molecular Fluorophores from Chitosan Assisted with Cellulase for Cell Nucleus Imaging and Photosensitive Printing

In this study, a facile and green route was designed using hydrolyzed chitosan as precursor to fabricate superior molecular state fluorophores based on the Maillard reaction (MR). The fluorophores exhibit excitation-dependent photoluminescence behavior, emitting multicolor fluorescence. The CCK-8 assay shows the low cytotoxicity of the Maillard reaction products (MRPs). The properties of the MRPs were associated with the molecular size. The high-molecular-weight fraction (>10 kDa) contains a large quantity of brown products which cause fluorescence quenching by an inner-filter effect. The medium-molecular-weight fraction (5–10 kDa) is able to permeate into the living cells and specifically stains the nucleus for stable imaging. The low-molecular-weight fraction (<3 kDa) possesses a dramatically high quantum yield of 30.4% and excellent ion stability, as well as a pH-responsive property; it can serve as a potential pH sensor and fluorescent ink for photosensitive printing. The potential applications of MRPs fractions in different fields are proposed, significantly improving the utilization efficiency. Additionally, this work provides a new perspective on designing and preparing large-scale metal-free fluorescence materials with high safety and good water solubility in a green and mild manner.

Correlation of chemically irreversible fouling with organic constituents of feed water during membrane filtration

Nowadays, fouling remains a huge obstacle for the application of membrane technology. Chemically irreversible fouling (CIF) is not cancelled by chemical cleaning (CC) and can cause long-term permeability loss. However, the characteristics of CIF foulants have not been systematically investigated yet. In this work, a pilot-scale long-term microfiltration process for surface water treatment with periodic CC was carried out to identify the nature of CIF foulants. A chemically irreversible fouling index (CIFI) approach was proposed to quantify CIF potential, and Pearson correlation analysis was further adopted to correlate CIFI with the organic constituents of feed water in terms of hydrophilicity/ hydrophobicity, molecular weight (MW) distribution and fluorophores. It was found that CIFI was significantly correlated to neutral hydrophilic fractions (R2 = 0.728, p-value = 0.007) and MW<1 kDa components (R2 = 0.749, p-value = 0.006), whereas no reliable correlation could be established of CIFI with fluorescent substances (humic-like or protein-like substances). Further, we found polysaccharides (PS) had a strong linear correlation with CIFI (R2 = 0.939, p = 0.0008) and the result was also supported by the detection of Fourier transform infrared spectroscopy. To sum up, the organics consisting of low MW (<1 kDa) and neutral hydrophilic PS-like constituents were mainly responsible to CIF. For the first time, we systematically reported the nature of CIF foulants in a low-pressure membrane filtration process.

Self-Enhanced Carbonized Polymer Dots for Selective Visualization of Lysosomes and Real-Time Apoptosis Monitoring.

SummaryProtons are highly related to cell viability during physiological and pathological processes. Developing new probes to monitor the pH variation could be extremely helpful to understand the viability of cells and the cell death study. Carbonized polymer dots (CPDs) are superior biocompatible and have been widely applied in bioimaging field. Herein, a new type of extreme-pH suitable CPDs was prepared from citric acid and o-phenylenediamine (CA/oPD-CPDs). Due to the co-existence of hydrophilic and hydrophobic groups, CA/oPD-CPDs tend to aggregate in neutral condition with a dramatic decrease of fluorescence, but disperse well in both acidic and alkaline conditions with brighter emission. This specialty enables them to selectively illuminate lysosomes in cells. Moreover, CA/oPD-CPDs in the cytoplasm could serve as a sustained probe to record intracellular pH variation during apoptosis. Furthermore, CA/oPD-CPDs present a continuous fluorescence increase upon 2-h laser irradiation in living cells, underscoring this imaging system for long-term biological recording.

Propylenedioxy Thiophene Donor to Achieve NIR-II Molecular Fluorophores with Enhanced Brightness

Developing molecular fluorophores with high brightness is of considerable importance to achieve superior biological imaging quality in the second near-infrared (NIR-II) window. It has been demonstr...

A molecular fluorophore in citric acid/ethylenediamine carbon dots identified and quantified by multinuclear solid-state nuclear magnetic resonance.

The composition of fluorescent polymer nanoparticles, commonly referred to as carbon dots, synthesized by microwave-assisted reaction of citric acid and ethylenediamine was investigated by 13 C, 13 C{1 H}, 1 H─13 C, 13 C{14 N}, and 15 N solid-state nuclear magnetic resonance (NMR) experiments. 13 C NMR with spectral editing provided no evidence for significant condensed aromatic or diamondoid carbon phases. 15 N NMR showed that the nanoparticle matrix has been polymerized by amide and some imide formation. Five small, resolved 13 C NMR peaks, including an unusual ═CH signal at 84 ppm (1 H chemical shift of 5.8 ppm) and ═CN2 at 155 ppm, and two distinctive 15 N NMR resonances near 80 and 160 ppm proved the presence of 5-oxo-1,2,3,5-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid (IPCA) or its derivatives. This molecular fluorophore with conjugated double bonds, formed by a double cyclization reaction of citric acid and ethylenediamine as first shown by Y. Song, B. Yang, and coworkers in 2015, accounts for the fluorescence of the carbon dots. Cross-peaks in a 1 H─13 C HETCOR spectrum with brief 1 H spin diffusion proved that IPCA is finely dispersed in the polyamide matrix. From quantitative 13 C and 15 N NMR spectra, a high concentration (18 ± 2 wt%) of IPCA in the carbon dots was determined. A pronounced gradient in 13 C chemical-shift perturbations and peak widths, with the broadest lines near the COO group of IPCA, indicated at least partial transformation of the carboxylic acid of IPCA by amide or ester formation.

Carbon dots-inspired fluorescent cyclodextrins: competitive supramolecular "off-on" (bio)sensors.

Chromophore-appended cyclodextrins combine the supramolecular loading capabilities of cyclodextrins (CDs) with the optical properties of the affixed chromophores. Among fluorescent materials, carbon dots (CNDs) are attractive and the feasibility of CND-appended CDs as sensors has been demonstrated by different authors. However, CNDs are intrinsically heterogeneous materials and their ulterior functionalization yields hybrid composites that are not well defined in terms of structure and composition. Inspired by the fluorescence properties of 5-oxo-1,2,3,5-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid (IPCA), the most paradigmatic of the molecular fluorophores detected in CNDs, herein we report two highly efficient synthetic chemical strategies for the preparation of IPCA-appended CDs that behave as CND-based CD "turn off-on" biosensors suitable for the analysis of cholesterol and β-galactosidase activity. We have deconstructed the CND-CD systems to demonstrate that (i) the role of CNDs is limited to acting as a support for the molecular fluorophores produced during their synthesis and (ii) the molecular fluorophores suffice for the determination of the enzymatic activity based on the quenching by p-nitrophenol as a sacrificial quencher.

Molecular engineering of a high quantum yield NIR-II molecular fluorophore with aggregation-induced emission (AIE) characteristics for in vivo imaging.

NIR-II biological imaging (1000-1700 nm) has shown promise for deep tissue penetration, high spatial resolution, and low background noise. Among all the NIR-II probes, organic probes particularly attract huge attention due to their excellent stability and biocompatibility, which have the most potential for clinical translation. However, most previously reported organic NIR-II fluorescent agents often suffer from low quantum yields in aqueous solution. Herein, we developed a novel D-π-A-π-D-type NIR II chromophore XA1 with AIE characteristics based on a new design strategy for NIR-II AIE fluorophores. Owing to their intrinsic aggregation-induced emission enhancement nature, the formulated XA1 NPs show a high fluorescence quantum yield up to 14.8%, which is higher than those of most previously reported organic NIR-II fluorophores. Based on the XA1 NPs, noninvasive imaging of limb and cerebral vessels is achieved with a high signal-to-background ratio and deep penetration. Furthermore, the XA1 NPs can be used as good contrast agents for high resolution imaging of blood vessels of tumors and precise detection of tumors based on the EPR mechanism. Collectively, our work demonstrated a novel strategy for designing and manufacturing NIR-II fluorophores with AIE characteristics and proved that XA1 NPs are highly promising NIR-II probes for biomedical imaging under physiological and pathological conditions.

Identification of Molecular Fluorophore as a Component of Carbon Dots able to Induce Gelation in a Fluorescent Multivalent-Metal-Ion-Free Alginate Hydrogel

We introduce a simple approach to fabricate fluorescent multivalent metal ion-free alginate hydrogels, which can be produced using carbon dots accessible from natural sources (citric acid and L-cysteine). Molecular fluorophore 5-oxo-2,3-dihydro-5H-[1,3]-thiazolo[3,2-a] pyridine-3,7-dicarboxylic acid (TPDCA), which is formed during the synthesis of carbon dots, is identified as a key segment for the crosslinking of hydrogels. The crosslinking happens through dynamic complexation of carboxylic acid groups of TPDCA and alginate cages along with sodium ions. The TPDCA derived hydrogels are investigated regarding to their thermal, rheological and optical properties, and found to exhibit characteristic fluorescence of this aggregated molecular fluorophore. Moreover, gradient hydrogels with tunable mechanical and optical properties and controlled release are obtained upon immersion of the hydrogel reactors in solutions of divalent metal ions (Ca2+, Cu2+, and Ni2+) with a higher affinity to alginate.

Gel electrophoresis separation and origins of light emission in fluorophores prepared from citric acid and ethylenediamine

We investigated light emission of hydrothermally treated citric acid and ethylenediamine (EDA) with various precursor ratios using gel-electrophoresis. We show that this relatively simple approach can deliver significant insights into the origins of photoluminescence. We found that products of the synthesis consist of both positively and negatively charged species and exhibit large dispersion in electrophoretic mobility (i.e. charge-to-size ratio). We observed that despite the large dispersion of the reaction products the blue light emission is confined to discrete bands clearly identifiable in the gel. We demonstrate clear evidence that this emission originates from the negatively charged light molecular fraction with the highest mobility which shows no excitation-dependent light emission. This molecular fluorophore exhibits spectral characteristics similar to previously reported 1,2,3,5-tetrahydro-5-oxo-imidazo[1,2-a]pyridine-7-carboxylic acid (IPCA). Secondary gel electrophoresis run performed on the bands extracted from the first run indicates that no further separation takes place. On the basis of our experimental results, we conclude that relatively stable binding exists between IPCA and EDA-derived product. Thus, the products of the reaction contain IPCA both in molecular form and in complexes with EDA-derived products. We conclude that excitation-dependent emission is related to the fluorophore binding to the positively charged EDA-derived products with a positive charge.

Blue and green luminescent carbon nanodots from controllable fuel-rich flame reactors

The continuous synthesis in controlled gas flame reactors is here demonstrated as a very effective approach for the direct and easy production of structurally reproducible carbon nanodots. In this work, the design of a simple deposition system, inserted into the reactor, is introduced. A controlled flame reactor is employed in the present investigation. The system was optimized for the production of carbon nanoparticles including fluorescent nanocarbons. Blue and green fluorescent carbon could be easily separated from the carbon nanoparticles by extraction with organic solvents and characterized by advanced chemical (size exclusion chromatography and mass spectrometry) and spectroscopic analysis. The blue fluorescent carbon comprised a mixture of molecular fluorophores and aromatic domains; the green fluorescent carbon was composed of aromatic domains (10–20 aromatic condensed rings), bonded and/or turbostratically stacked together. The green-fluorescent carbon nanodots produced in the flame reactor were insoluble in water but soluble in N-methylpyrrolidinone and showed excitation-independent luminescence. These results provide insights for a simple and controlled synthesis of carbon nanodots with specific and versatile features, which is a promising pathway for their use in quite different applicative sectors of bioimaging.

Self-illumination of Carbon Dots by Bioluminescence Resonance Energy Transfer

Carbon-dots (CDs), the emerging fluorescent nanoparticles, show special multicolor properties, chemical stability, and biocompatibility, and are considered as the new and advanced imaging probe in replacement of molecular fluorophores and semiconductor quantum dots. However, the requirement of external high power light source limits the application of fluorescent nanomaterials in bio-imaging. The present study aims to take advantage of bioluminescence resonance energy transfer mechanism (BRET) in creating self-illuminating C-dots. Renilla luciferase (Rluc) is chosen as the BRET donor molecule. Conjugation of Renilla luciferase and C-dots is necessary to keep their distance close for energy transfer. The optimal condition for achieving BRET is investigated by studying the effects of different factors on the performance of BRET, including the type of conjugation, concentration of carbon dots, and conjugation time. The linear relationship of BRET efficiency as a function of the amount of C-dots in the range of 0.20–0.80 mg/mL is observed. The self-illuminating carbon dots could be applied in bioimaging avoiding the tissue damage from the external high power light source.

Carbon dots with molecular fluorescence and their application as a \u201cturn-off\u201d fluorescent probe for ferricyanide detection

Highly fluorescent carbon dots (CDs) exhibiting molecular fluorescence were synthesized and successfully used for sensing ferricyanide based on fluorescence quenching. We conducted dialysis to purify the CDs and found that the dialysate is also fluorescent. From the mass spectra and quantum yield analyses of the dialysate, it is demonstrated that molecular fluorophores were also synthesized during the synthesis of CDs. By the comparison of fluorescence spectra between CDs and dialysate, it is established that the fluorescence emission of CDs partly originates from fluorophores that are attached to CDs’ surface. The fluorescence quenching caused by ferricyanide is proved to be the overlap of absorption spectra between ferricyanide and CDs. The changes of the absorbance and fluorescence spectra are combined to enhance the detection sensitivity, and the limit of detection is calculated to be 1.7 μM. A good linear response of fluorescence-absorbance combined sensing toward ferricyanide is achieved in the range of 5–100 µM. This method is highly selective to ferricyanide among other common cations and anions, and it is also successfully applied in detecting ferricyanide in real water samples.