Ratiometric Dual Emission Research Articles

Design, synthesis and application of dual-channel fluorescent probes for ratiometric detection of HClO and H2S based on phenothiazine coumarins

The ubiquity of diverse material entities in environmental matrices renders the deployment of unifunctional fluorescent indicators inadequate. Consequently, this study introduces a ratiometric dual-emission fluorescent sensor (Probe CP), synthesized by conjugating phenothiazine coumarin to hydroxycoumarin through a piperazine linker for concurrent detection of HClO and H2S. Upon interaction with HClO, the phenothiazine unit's sulfur atom undergoes oxidation to sulfoxide, facilitating a shift from red to green fluorescence in a ratiometric manner. Concurrently, at the opposite terminus of Probe CP, 2,4-dinitroanisole serves as the reactive moiety for H2S recognition; it restores the blue emission characteristic of 7-hydroxycoumarin while maintaining the red fluorescence emanating from phenothiazine coumarin as an internal standard for ratio-based assessment. Exhibiting elevated specificity and sensitivity coupled with minimal detection thresholds (0.0506 μM for HClO and 1.7292 μM for H2S) alongside rapid equilibration periods (3 min for HClO and half an hour for H2S), this sensor was efficaciously employed in cellular environments and within zebrafish models as well as imaging applications pertaining to alcohol-induced hepatic injury in murine subjects.

Engineering Tunable Ratiometric Dual Emission in Single Emitter-based Amorphous Systems.

Molecular emitters with multi-emissive properties are in high demand in numerous fields, while these properties basically depend on specific molecular conformation and packing. For amorphous systems, special molecular arrangement is unnecessary, but it remains challenging to achieve such luminescent behaviors. Herein, we present a general strategy that takes advantage of molecular rigidity and S1 -T1 energy gap balance for emitter design, which enables fluorescence-phosphorescence dual-emission properties in various solid forms, whether crystalline or amorphous. Subsequently, the amorphism of the emitters based polymethyl methacrylate films endowed an in situ regulation of the dual-emissive characteristics. With the ratiometric regulation of phosphorescence by external stimuli and stable fluorescence as internal reference, highly controllable luminescent color tuning (yellow to blue including white emission) was achieved. There properties together with a persistent luminous behavior is of benefit for an irreplaceable set of optical information combination, featuring an ultrahigh-security anti-counterfeiting ability. Our research introduces a concept of eliminating the crystal-form and molecular-conformational dependence of complex luminescent properties through emitter molecular design. This has profound implications for the development of functional materials.

Engineering Tunable Ratiometric Dual Emission in Single Emitter‐based Amorphous Systems

AbstractMolecular emitters with multi‐emissive properties are in high demand in numerous fields, while these properties basically depend on specific molecular conformation and packing. For amorphous systems, special molecular arrangement is unnecessary, but it remains challenging to achieve such luminescent behaviors. Herein, we present a general strategy that takes advantage of molecular rigidity and S1‐T1 energy gap balance for emitter design, which enables fluorescence–phosphorescence dual‐emission properties in various solid forms, whether crystalline or amorphous. Subsequently, the amorphism of the emitters based polymethyl methacrylate films endowed an in situ regulation of the dual‐emissive characteristics. With the ratiometric regulation of phosphorescence by external stimuli and stable fluorescence as internal reference, highly controllable luminescent color tuning (yellow to blue including white emission) was achieved. There properties together with a persistent luminous behavior is of benefit for an irreplaceable set of optical information combination, featuring an ultrahigh‐security anti‐counterfeiting ability. Our research introduces a concept of eliminating the crystal‐form and molecular‐conformational dependence of complex luminescent properties through emitter molecular design. This has profound implications for the development of functional materials.

Ratiometric dual-emission carbon dots coupled with smartphone for visual quantification of Co2+ and EDTA and biological sensing

Ratiometric fluorescence analysis provides the feasibility for visual quantitative sensing by presenting continuous color changes. Herein, dual-emission carbon dots (D-CDs) have been developed for ratiometric recognition of Co2+ and EDTA, along with successive fluorescence variation of yellow, purplish-pink, purple, light blue, and dark blue. D-CDs have been synthesized by microwave-assisted heating of tetracycline and manifest dual emissions at 562 nm and 610 nm under 450 nm excitation. Intriguingly, the emissions at 562 nm and 610 nm are quenched while a ratio rising emission emerges at 482 nm upon introducing Co2+, which displays ratiometric fluorescence emission characteristics (I482/I562) in the range of 0.25–400 µM with significant fluorescence varying from yellow, purplish-pink, purple, light blue, to dark blue. Subsequent addition of EDTA could ratiometric recover the fluorescence of D-CDs in the range of 50–130 µM with the fluorescence returning from dark blue, light blue, purple, purplish-pink, to yellow. More crucially, integrating continuous color changes and color recognizer APP in the smartphone, visual quantification of Co2+ and EDTA can be realized based on the ratio of blue value and red value (B/R) with linear ranges of 0–400 µM and 0–300 µM, respectively. Furthermore, proposed D-CDs could be extended as a visual biosensing platform for tracking Co2+ and EDTA in living cells, which have potentialapplication prospect in biosystem.

A New Spectral Shift-Based Method to Characterize Molecular Interactions.

There are many fluorescence-based applications that can be used to characterize molecular interactions. However, available methods often depend on site-specific labeling techniques or binding-induced changes in conformation or size of the probed target molecule. To overcome these limitations, we applied a ratiometric dual-emission approach that quantifies ligand-induced spectral shifts with sub-nanometer sensitivity. The use of environment-sensitive near-infrared dyes with the method we describe enables affinity measurements and thermodynamic characterization without the explicit need for site-specific labeling or ligand-induced conformational changes. We demonstrate that in-solution spectral shift measurements enable precise characterization of molecular interactions for a variety of biomolecules, including proteins, antibodies, and nucleic acids. Thereby, the described method is not limited to a subset of molecules since even the most challenging samples of research and drug discovery projects like membrane proteins and intrinsically disordered proteins can be analyzed.

Ratiometric dual-emission of Rhodamine-B grafted carbon dots for full-range solvent components detection

Quick and visual detection of component contents, such as water, in a mixed solvent is important for many practical applications, and a full range detection is especially preferred. In this work, a carbon dots based ratiometric fluorescent sensor was synthesized by grafting fluorescent group (Rhodamine B, RhB) on carbon dots, and the dual emission peaks exhibited a linear ratiometric response with the change of polarity and hydrogen bond of Solvent Hansen solubility parameters. This responsive behavior is attributed to surface state photoluminescence mechanisms, and has been used for the quantitative detection of water content in ethanol with an excellent linear relationship (R2 = 0.996), a low detection limit (0.2%), and a full detection range (0–100%). Furthermore, a paper-based ratiometric fluorescence sensing strip is also demonstrated, which exhibits good storage stability and sensitivity. This study suggests that RhB grafted carbon dots could be feasibly and effectively used as ratiometric fluorescent sensors for solvent content detection.

Ratiometric fluorescent sensing of ethanol based on copper nanoclusters with tunable dual emission

Copper nanoclusters (Cu NCs) have attracted a surge of interest in fluorescent sensors as their outstanding physicochemical and optical properties. However, most of the reports have focused on single-signal fluorescent sensors, which are susceptible to background interferences and affect accuracy of the results. Herein, we constructed a facile ratiometric fluorescent sensor for monitoring ethanol based on Cu NCs with tunable dual emission. Polyvinylpyrrolidone (PVP)-modified Cu NCs were simply prepared in water, which exhibit ratiometric dual emission, including a strong green emission at 520 nm and a weak blue emission at 450 nm. The PVP-Cu NCs in water with strong green emission display monodisperse state due to the formation of hydration shell around Cu NCs. In ethanol where the hydration shell is destructed, Cu NCs tend to aggregate and show strong blue emission. This emission shift might attribute to the enhancement of Cu–Cu metallophilic interaction with the aggregation of Cu NCs, which induces the excited-state level increasing. Thus, a ratiometric fluorescent probe for ethanol based on the PVP-Cu NCs is fabricated, which possesses rapid response (<1 min), and realize full-range detection from 0 to 100%. In addition, this ratiometric probe is successfully applied to determine the alcohol strength of alcohol beverages, demonstrating the great potential in practical application.

Supramolecular Assembly of An Organoplatinum(II) Complex with Ratiometric Dual Emission for Two-Photon Bioimaging.

The organoplatinum(II) complex [Pt(C^N^N)(Cl)] (C^N^N=5,6-diphenyl-2,2'-bipyridine, Pt1) can assemble into nanoaggregates via π-π stacking and complementary hydrogen bonds, rather than Pt-Pt interactions. Pt1 exhibits ratiometric dual emission, including rare blue emission (λem =445 nm) and assembly-induced yellow emission (λem =573 nm), under one- and two-photon excitation. Pt1 displays blue emission in cells with an intact membrane due to its low cellular uptake. In cells where the membrane is disrupted, uptake of the complex is increased and at higher concentrations yellow emission is observed. The ratio of yellow to blue emission shows a linear relationship to the loss of cell membrane integrity. Pt1 is, to our knowledge, the first example of an assembly-induced two-photon ratiometric dual emission organoplatinum complex. The excellent and unique characteristics of the complex enabled its use for the tracking of cell apoptosis, necrosis, and the inflammation process in zebrafish.

Supramolecular Assembly of An Organoplatinum(II) Complex with Ratiometric Dual Emission for Two‐Photon Bioimaging

AbstractThe organoplatinum(II) complex [Pt(C^N^N)(Cl)] (C^N^N=5,6‐diphenyl‐2,2′‐bipyridine, Pt1) can assemble into nanoaggregates via π–π stacking and complementary hydrogen bonds, rather than Pt–Pt interactions. Pt1 exhibits ratiometric dual emission, including rare blue emission (λem=445 nm) and assembly‐induced yellow emission (λem=573 nm), under one‐ and two‐photon excitation. Pt1 displays blue emission in cells with an intact membrane due to its low cellular uptake. In cells where the membrane is disrupted, uptake of the complex is increased and at higher concentrations yellow emission is observed. The ratio of yellow to blue emission shows a linear relationship to the loss of cell membrane integrity. Pt1 is, to our knowledge, the first example of an assembly‐induced two‐photon ratiometric dual emission organoplatinum complex. The excellent and unique characteristics of the complex enabled its use for the tracking of cell apoptosis, necrosis, and the inflammation process in zebrafish.

An excitation-dependent ratiometric dual-emission strategy for the large-scale enhancement of fluorescent tint control

An alternative and convenient strategy for preparing carbon dots (CDs) with multicolor and dual-emission fluorescence is described. For this dual-emission characteristic, the short-wavelength emission reveals unique excitation-dependent fluorescence behavior, during which the long-wavelength emission remains unshifted regardless of the excitation. Consequently, such excitation-dependent ratiometric dual emission can be applied into a fluorescent tint control of this material between the cold and warm white-light regions. This unique property allows the CDs to be further translated into film sheets for visual detection of the irradiation source, and to also be conjugated with calf thymus DNA for multichannel bioimaging. These results offer new insights for the development of easy-to-handle techniques for material luminescent color tuning.

A novel ratiometric dual-emission fluorescence magnetic nanohybrid for HIgG immunoassay

The proposed ratiometric dual-emission (carbon dots and QDs) fluorescence magnetic nanohybrid displayed excellent analytical performance for the detection of HIgG.

Modulation of a Photoswitchable Dual-Color Quantum Dot containing a Photochromic FRET Acceptor and an Internal Standard

Photoswitchable semiconductor nanoparticles, quantum dots (QDs), couple the advantages of conventional QDs with the ability to reversibly modulate the QD emission, thereby improving signal detection by rejection of background signals. Using a simple coating methodology with polymers incorporating a diheteroarylethene photochromic FRET acceptor as well as a spectrally distinct organic fluorophore, photoswitchable QDs were prepared that are small, biocompatible, and feature ratiometric dual emission. With programmed irradiation, the fluorescence intensity ratio can be modified by up to ∼100%.

Multicompartment polymer nanostructures with ratiometric dual-emission pH-sensitivity.

Pyrazine-labeled multicompartment nanostructures are shown to exhibit enhanced pH-responsive blue-shifted fluorescence emission intensities compared to their simpler core-shell spherical analogs. An amphiphilic linear triblock terpolymer of ethylene oxide, N-acryloxysuccinimide, and styrene, PEO(45)-b-PNAS(105)-b-PS(45), which lacks significant incompatibility for the hydrophobic block segments and undergoes gradual hydrolysis of the NAS units, underwent supramolecular assembly in mixtures of organic solvent and water to afford multicompartment micelles (MCMs) with a narrow size distribution. The assembly process was followed over time and found to evolve from individual polymer nanodroplets containing internally phase segregated domains, of increasing definition, and ultimately to dissociate into discrete micelles. Upon covalent cross-linking of the MCMs with pH-insensitive pyrazine-based diamino cross-linkers, pH-responsive, photonic multicompartment nanostructures (MCNs) were produced. These MCNs exhibited significant enhancement of overall structural stability, in comparison with the MCMs, and internal structural tunability through the cross-linking chemistry. Meanwhile, the complex compartmentalized morphology exerted unique pH-responsive fluorescence dual-emission properties, indicating promise in ratiometric pH-sensing applications.

Green Fluorescent Protein Variants as Ratiometric Dual Emission pH Sensors. 3. Temperature Dependence of Proton Transfer

In parts 1 and 2 of this series [Hanson, G. T., McAnaney, T. B., Park, E. S., Rendell, M. E. P., Yarbrough, D. K., Chu, S. Y., Xi, L. X., Boxer, S. G., Montrose, M. H., and Remington, S. J. (2002) Biochemistry 41, 15477-15488; McAnaney, T. B., Park, E. S., Hanson, G. T., Remington, S. J., and Boxer, S. G. (2002) Biochemistry 41, 15489-15494], we described the structure, excited-state dynamics, and applications of pH-sensitive, ratiometric dual emission green fluorescent protein (deGFP) variants with fluorescence emission that is modulated between blue (lambda(max) approximately equal 465 nm) and green (lambda(max) approximately equal 515 nm) depending on the pH of the bulk solvent. In this paper, we consider the energetic origin of the dual emission properties of these GFP variants by examining the temperature dependence of the steady-state absorption and fluorescence emission. In most cases, the quantum yield of the green emission decreased as the temperature was lowered, indicating that the excited-state proton transfer (ESPT) which produces the green emitting form is an activated process. The activation energies of ESPT, determined by modeling the quantum yields of both blue and green emissions between 260 and 298 K in the context of a simple photocycle, were found to be larger at low pH than at high pH. These results indicate that the ratiometric dual emission properties of deGFP mutants are due to this pH-sensitive ESPT rate, combined with a modulation of the ground-state neutral and anionic chromophore populations with pH. The time-resolved fluorescence of one of the deGFP mutants was studied in detail. The time-resolved emission spectra of this mutant are the first ultrafast spectra obtained for a GFP. These spectra demonstrate that the rising kinetics for green emission, considered a hallmark of ESPT, is the sum of the contribution from both the neutral and intermediate anionic forms of the chromophore at the probe wavelength and may not be observed in all mutants that undergo ESPT, depending on the relative contributions of the two forms.

1,3-Diphenyl-1H-pyrazolo[3,4-b]quinoline: a versatile fluorophore for the design of brightly emissive molecular sensors.

[structure: see text] The 1,3-diphenyl-1H-pyrazolo[3,4-b]-quinoline chromophore is a versatile building block for the construction of brightly fluorescent molecular sensors. Facile synthetic procedures allow integration of the chromophore into fluorophore-spacer-receptor systems as well as fluoroionophores operating via intramolecular charge transfer. Whereas the former photoinduced electron-transfer probes show strong analyte-induced fluorescence enhancement, the latter exhibit bright ratiometric dual emission. Employing prototype macrocyclic receptors, the favorable signaling features for metal ion recognition are demonstrated.

Rapid, simple and sensitive microassay for skeletal muscle homogenates in the functional assessment of the Ca-release channel of sarcoplasmic reticulum: application to diagnosis of susceptibility to malignant hyperthermia.

A microassay is demonstrated for functional characterization of the Ca(2+)-release channel (CRC) of sarcoplasmic reticulum (SR) of skeletal muscle using swine with susceptibility to malignant hyperthermia (MH). Diluted muscle homogenates, indo-1 and ratiometric dual-emission spectrofluorometry are used to monitor Ca(2+)-lowering activity in real-time in the presence and absence of ryanodine at exposures that open and close the CRC. Reactions are initiated with 50 microM CaCl2 to raise ionized Ca2+ concentration near 1 microM and MgATP to activate the Ca(2+)-ATPase pump. Oxalate is included to precipitate Ca2+ within the SR. The assay requires less than 30 mg muscle, which may be cryopreserved, and is completed within 20 min of thawing the tissue. Maximum SR Ca(2+)-ATPase pumping and CRC activities, degree of CRC activation, and Ca(2+)-buffering capacity can be determined. Using this assay we studied muscle from MH-susceptible swine and demonstrated that whereas maximal Ca(2+)-ATPase pumping and CRC activities are normal, the CRC activity after addition of a bolus of Ca2+ is 50% greater in heterozygotes and 100% greater in homozygotes for the MH mutation. Hypersensitivity to CRC agonists, such as caffeine, and an associated hyposensitivity to CRC antagonists such as Mg2+ is also demonstrated. Genotypes for the MH mutation site can be discriminated from each other by determining Ca(2+)-lowering activities and the effect of ryanodine on them.

Lymphocyte calcium extrusion: kinetic and thermodynamic measurements using ratiometric dual-emission spectrofluorometry

A method is described for monitoring intracellular ionized calcium (Ca2+) and determining kinetic and thermodynamic parameters of Ca2(+)-extrusion from intact lymphocytes. The method uses ratiometric spectrofluorometry and the fluorescent Ca2+ dye indo-1. Lymphocytes were loaded with calcium and placed in a low calcium medium. A novel formula for calculation of intracellular Ca2+ that corrects for background fluorescence and fluorescence quenching was used. Calcium extrusion resulted in exponential decrease in cytoplasmic Ca2+ with a rate constant of 0.031 +/- 0.003 sec-1, maximal rate of 23 +/- 7 nM/sec, dissociation constant of 366 +/- 63 nM, Hill coefficient of 2.3 +/- 0.4, Q10 of 2.58 +/- 0.28, and activation energy of 18.3 Kcal/mol. This method should allow for characterization of the Ca2(+)-extrusion system of lymphocytes and may be applicable to other blood cell types.

Calcium sequestration by isolated sarcoplasmic reticulum: real-time monitoring using ratiometric dual-emission spectrofluorometry and the fluorescent calcium-binding dye indo-1

This study demonstrates a simple, rapid, and reproducible microassay for real-time monitoring of Ca2(+)-sequestration by isolated sarcoplasmic reticulum (SR) using ratiometric dual-emission spectrofluorometry and the fluorescent calcium-binding dye indo-1. The SR membranes were isolated by differential centrifugation and suspended in a medium including Ca2+, indo-1, ATP and oxalate. As Ca2+ was sequestered by SR, Ca2(+)-bound indo-1 fluorescence decreased equivalently but reciprocally to the increase in Ca2(+)-free indo-1 fluorescence. The kinetic and thermodynamic properties of Ca2(+)-transport measured fluorometrically were similar to those measured radiometrically by 45Ca2+, with the exception that the former monitors changes in free Ca2+ whereas the latter monitors total Ca2+. An estimate of the maximal rate of change in total Ca2+ could be made by multiplying the maximal rate of change in free Ca2+ by the ratio of initial total Ca2+ to free Ca2+ concentration.