Fluorescein Dianion Research Articles

Blue-Shifted and Broadened Fluorescence Enhancement by Visible and Mode-Selective Infrared Double Excitations.

Mode-selective vibrational excitations to modify the electronic states of fluorescein dianion in methanol solutions are carried out with a femtosecond visible pulse synchronized with a tunable high-power, narrow-band picosecond infrared (IR) pulse. In this work, simultaneous intensity enhancement, peak blueshift, and line width broadening of fluorescence are observed in the visible/IR double resonance experiments. Comprehensive investigations on the modulation mechanism with scanning the vibrational excitation frequencies, tuning the time delay between the two excitation pulses, theoretical calculations, and nonlinear and linear spectroscopic measurements suggest that the fluorescence intensity enhancement is caused by the increase of the Franck-Condon factor induced by the vibrational excitations at the electronic ground state. Various enhancement effects are observed as vibrations initially excited by the IR photons relax to populate the vibrational modes of lower frequencies. The peak blueshift and line width broadening are caused by both increasing the Franck-Condon factors among different subensembles because of IR pre-excitation and the long-lived processes induced by the initial IR excitation. The results demonstrate that the fluorescence from the visible/IR double resonance experiments is not a simple sum frequency effect, and vibrational relaxations can produce profound effects modifying luminescence.

Unraveling the mystery of solvation-dependent fluorescence of fluorescein dianion using computational study.

Fluorescein, one of the brightest fluorescent dye molecules, is a widely used fluorophore for various applications from biomedicine to industry. The dianionic form of fluorescein is responsible for its high fluorescence quantum yield. Interestingly, the molecule was found to be nonfluorescent in the gas phase. This characteristic is attributed to the photodetachment process, which out-competes the fluorescence emission in the gas phase. In this work, we show that the calculated vertical and adiabatic detachment energies of fluorescein dianion in the gas and solvent phases account for the drastic differences observed in their fluorescence characteristics. The functional dependence of these detachment energies on the dianion's microsolvation was systematically investigated. The performance of different solvent models was also assessed. The higher thermodynamic stability of fluorescein dianion over the monoanion doublet in the solvent phase plays a crucial role in quenching photodetachment and activating the radiative channel with a high fluorescence quantum yield.

Functioning of a Fluorescein pH-Probe in Aqueous Media: Impact of Temperature and Viscosity.

In this work, we considered the influence of viscogenic agents (glycerol, sucrose) as well as the temperature on the fluorescent characteristics of fluorescein at pH 6.5 in order to describe the acid-base status of local environment in terms of a spectrally detectable dianion-anion equilibrium. The protolytic equilibrium of fluorescein was found to depend on the solvent viscosity in a complex way. Whereas in the presence of sucrose the ratiometric signal of fluorescein (I488/I435) remains rather unchanged, the addition of glycerol (up to 40% w/w) results in the increase of the signal (up to 19%), that can be attributed to the different mechanisms of cosolvents effects on dye molecules in the ground state. Molecular dynamics of the dye in the presence of glycerol and sucrose revealed that the cosolvents preferentially interact with fluorescein monoanion and dianion, displacing water molecules from the local environment which in turn reduces the average number of the hydrogen bonds between xanthene ring of the dye and water molecules. The ratiometric signal demonstrates linear growth with the temperature in the range of 10-80 °C regardless of the presence of viscogenic agents. A linear correlation between the temperature sensitivity of the ratiometric signal and the change in the molar enthalpy of the proton dissociation reaction in buffer and viscous media was determined.

Unraveling the decarboxylation dynamics of the fluorescein dianion with fragment action spectroscopy

The decarboxylation dynamics of the doubly deprotonated fluorescein dianion, Fl2-, are investigated by recording fragment action spectra for the anion, Fl-, and its decarboxylated analog, Fl-CO2-, using a new reflectron secondary mass spectrometer. The formation of the anion, Fl-, is directly investigated by photoelectron imaging. The Fl- and Fl-CO2- action spectra indicate that, for λ < 400nm, one-photon dissociative photodetachment, i.e., simultaneous decarboxylation and electron loss, competes with photodetachment, whereas for λ > 400nm, decarboxylation only proceeds following electron loss via a sequential two-photon process. The primary decarboxylation pathway is the ready loss of CO2 from the relatively short-lived intermediate excited state, Fl-[D1], which is formed by electron loss from the dianion via resonant tunneling through the repulsive Coloumb barrier associated with a high-lying excited dianion state, Fl2-[S2].

Hybrid, dual visible and near-infrared fluorescence emission of (6,5) single-walled carbon nanotubes modified with fluorescein through aryl diazonium salt chemistry

The aryl diazonium salt chemistry offers enhancement of near-infrared (NIR) emission of single-walled carbon nanotubes (SWCNTs), although, the attachment of functional molecules which could bring hybrid properties through the process is underdeveloped. In this work, we utilize aryl diazonium salt of fluorescein to create sp 3 defects on (6,5) SWCNTs. We study the influence of pH on the grafting process identifying that pH 5–6 is necessary for a successful reaction. The fluorescein-modified (6,5) SWCNTs (F-(6,5) SWCNTs) exhibit red-shifted E 11* emission in the NIR region attributed to luminescent sp 3 defects, but also visible (Vis) fluorescence at 515 nm from surface-attached fluorescein molecules. The fluorescence in both Vis and NIR regions of F-(6,5) SWCNTs exhibit strong pH-dependency associated with the dissociation of fluorescein molecules with an indication of photoinduced-electron transfer quenching the Vis emission of fluorescein dianion. The F-(6,5) SWCNTs could potentially be used for dual-channel medical imaging as indicated by our preliminary experiments. We hope that our research will encourage new, bold modifications of SWCNTs with functional molecules introducing new, unique hybrid properties.

Kasha's Rule and Koopmans' Correlations for Electron Tunnelling through Repulsive Coulomb Barriers in a Polyanion.

The long-range electronic structure of polyanions is defined by the repulsive Coulomb barrier (RCB). Excited states can decay by resonant electron tunnelling through RCBs, but such decay has not been observed for electronically excited states other than the first excited state, suggesting a Kasha-type rule for resonant electron tunnelling. Using action spectroscopy, photoelectron imaging, and computational chemistry, we show that the fluorescein dianion, Fl2–, partially decays through electron tunnelling from the S2 excited state, thus demonstrating anti-Kasha behavior, and that resonant electron tunnelling adheres to Koopmans’ correlations, thus disentangling different channels.

Ionic liquids for active photonics components fabrication

We report the new fluorescent polymerizable imidazolium-based ionic liquids for photonics application. We investigate two approaches: one ionic liquid (IL) contains the active fluorescein dianion as an intrinsic part of the polymerizable ionic liquid structure. The second liquid is based on the mixture of polymerizable ionic liquid with optically active europium(III) nitrate hydrate. We demonstrate the polymerization abilities of both liquids by means of electron beam in vacuum conditions. Furthermore, we measure the light emission properties of liquid and polymerized structures. The results reveal that IL with europium(III) nitrate hydrate yields emission spectrum with Eu3+ bands not only as a liquid but also as a polymerized solid microstructure, which is important for its application in active photonics components.

Fluorescence of aminofluoresceins as an indicative process allowing one to distinguish between micelles of cationic surfactants and micelle-like aggregates

Among the vast set of fluorescein derivatives, the double charged R2− anions of aminofluoresceins are known to exhibit only low quantum yields of fluorescence, . The value becomes as high as that of the fluorescein dianion when the lone electron pair of the amino group is involved in a covalent bond. According to Munkholm et al (1990 J. Am. Chem. Soc. 112 2608–12), a much smaller increase in the emission intensity can be observed in the presence of surfactant micelles. However, all these observations refer to aqueous or alcoholic solvents. In this paper, we show that in the non-hydrogen bond donor (or ‘aprotic’) solvents DMSO and acetone, the quantum yields, φ, of the 4′- (or 5′)-aminofluorescein R2− species amount to 61–67% and approach that of fluorescein (φ = 87%), whereas in water φ is only 0.6–0.8%. In glycerol, a solvent with an extremely high viscosity, the φ value is only 6–10%.We report on the enhancement of the fluorescence of the aminofluorescein dianions as an indicative process, which allows us to distinguish between the micelle-like aggregates of cationic dendrimers of low generation, common spherical surfactant micelles, and surfactant bilayers. Some of these colloidal aggregates partly restore the fluorescence of aminofluoresceins in aqueous media. By contrast, other positively charged micellar-like aggregates do not enhance the quantum yield of aminofluorescein R2− species. Results for several related systems, such as CTAB-coated SiO2 particles and reverse microemulsions, are briefly described, and the possible reasons for the observed phenomena are discussed.

Fluorescein-based chromo-fluorescent probe for zinc in aqueous solution: Spirolactam ring opened or closed?

A fluorescein derivative, sensor 1, was synthesized and characterized. It shows 50nm bathochromic shifts (from 346 to 400nm) with a change of solution color and a large increase in the fluorescence intensity upon binding zinc in 1:1 ethanol–hydroxyethyl piperazine ethanesulfonic acid buffer (v/v, pH 7.2) solution. It was demonstrated that changes are not due to the ring opening of the fluorescein spirolactam. The formation of 1–Zn2+ complex or fluorescein dianion adduct at higher pH was further confirmed by mass spectrometry. The fluorescein based molecular probe showed Zn2+ induced absorption and emission changes but does not undergo spirolactam ring opening (reversible) whereas ring opening (irreversible) and hydrolysis take place in alkaline pH region. Confocal microscopy experiments showed that 1 can be used for detection of Zn2+ levels (∼30μM) within living cells.

Solvatochromic and preferential solvation of fluorescein in some water-alcoholic mixed solvents

It is known that the hydrogen bonding has a significant effect on the spectral properties of fluorescein, however its relevance to solution behavior and preferential solvation of fluorescein is unclear in mixed solvents. In this work, absorption spectra of fluorescein dianion have been investigated in pure water, methanol, ethanol, 1-propanol, 2-propanol, dimethylsulfoxide, N,N-dimethylformamide, acetonitrile and in binary mixtures of water with the mentioned alcohols at 298.15K. Spectral changes were interpreted in terms of specific and nonspecific solute-solvent interactions. The energy of electronic transition in maximum absorption (ET) was calculated in each binary mixture. The extent and importance of each of solute–solvent interactions to ET were analyzed in the framework of the linear solvation energy relationships. Preferential solvation was detected as a nonideal behavior of ET curve respective to the analytical mole fraction of alcohols in all binary mixtures. The ET values were fitted to a solvent exchange model to calculate the preferential solvation parameters. The preference of fluorescein dianion to be solvated by one of the solvating species relative to others was discussed in terms of solvent–solvent and solute–solvent interactions.

Excited-State Proton Transfer of Fluorescein Anion as an Ionic Liquid Component

Fluorescent ionic liquids (FILs) incorporating the fluorescein anion have been prepared by anion exchange of the parent quaternary ammonium chloride (Quat(+)Cl(-)) ionic liquid. By controlling the molar ratio of fluorescein to Quat(+)Cl(-), ionic liquids incorporating different prototropic forms of fluorescein were prepared. The 1:1 molar ratio ionic liquid (FIL1) is essentially composed of monoanionic fluorescein, while dianionic fluorecein is predominant in the FIL with a 1:2 molar ratio (FIL2). The fluorescence excitation spectrum of FIL2 is markedly different from its absorption spectrum. Absorption features the fluorescein dianion, while the excitation spectrum is exclusively due to the monoanion. In FIL1, the absorption and excitation spectra are both characteristic of the monoanion. In both FILs, emission of the dianion is observed upon excitation of the monoanion. This unusual behavior is interpreted in the context of a fast deprotonation of the monoanion in the excited state. The presence of residual water in the ionic liquid is important for the proton transfer process. By lowering the pH of FIL1, the transient proton transfer is inhibited, and the emission of the monoanion could be observed. The FILs have completely different spectroscopic properties from solvated fluorescein in Quat(+)Cl(-), where the prototropic equilibrium is shifted toward the neutral forms.

One- and two-photon absorption of fluorescein dianion in water: A study using S-QM/MM methodology and ZINDO method

One- and two-photon absorption (1PA and 2PA) of fluorescein dianion (FSD) in water were studied using a combined and sequential Quantum Mechanics/Molecular Dynamics methodology. Different sets of 250 statistically relevant (uncorrelated) configurations composed by the solute and several solvent molecules were sampled from the classical simulation. On these configurations, the electronic properties were calculated a posteriori using the Zerner’s intermediate neglect of differential overlap (ZINDO) method. The linear and nonlinear absorption of FSD in water were calculated using discrete and explicit solvent models. In the largest case, the relevant configurations are composed by FSD and 47 explicit water molecules embedded in the electrostatic field of all remaining water molecules. Both INDO/CIS and INDO/CISD calculations were performed to study the absorption processes of FSD and the Sum-Over-States (SOS) model was used to describe the 2PA process. A semi-classical method for spectrum simulations was employed to simulate the 1PA and 2PA cross-section spectra of FSD in water. For comparison purposes, in the case of the 2PA process two approaches, the “full expression” and “resonant expression” methods, were employed to simulate the nonlinear spectrum. The last method assumes resonant conditions and on the computation point of view it represents an interesting option to study the 2PA process. The INDO/CI calculations give a satisfactory description of the 1PA spectrum of FSD and properly describe the unusual blue-shift of its first π→π* transition in water. In the case of 2PA, the introduction of doubly excited configuration interactions (INDO/CISD) has proven to be essential for an appropriate description of the process at the higher energy spectral region. It was observed that the solvent effects do not drastically change the cross-sections of both processes. The simulated 2PA cross-section spectrum provided by the “full expression” method presents a better definition of the bands which appear along the experimental spectrum than the one provided by the “resonant expression” method. However, both approaches provide similar description for the effect of the solvent environment on the 2PA process of FSD in water.

Universal Chemiluminescence Flow-Through Device Based on Directed Self-Assembly of Solid-State Organic Chromophores on Layered Double Hydroxide Matrix

In this work, a universal chemiluminescence (CL) flow-through device suitable for various CL resonance energy transfer (CRET) systems has been successfully fabricated. Highly efficient CRET in solid-state photoactive organic molecules can be achieved by assembling them on the surface of layered double hydroxides (LDHs). We attribute these observations to the suppression of the intermolecular π-π stacking interactions among aromatic rings and the improvement of molecular orientation and planarity in the LDH matrix, enabling a remarkable increase in fluorescence lifetime and quantum yield of organic molecules. Under optimal conditions, using peroxynitrous acid-fluorescein dianion (FLUD) as a model CRET system, trace FLUD (10 μM) was assembled on the surface of LDHs. Peroxynitrous acid/nitrite could be assayed in the range of 1.0-500 μM, and the detection limit for peroxynitrous acid/nitrite (S/N = 3) was 0.6 μM. This CL flow-through device exhibited operational stability, high reproducibility, and long lifetime. While LDHs were immobilized in a flow-through device in the absence of FLUD, the detection limit for peroxynitrous acid/nitrite was 100 μM. On the other hand, FLUD at the same concentration can not enhance the CL intensity of peroxynitrous acid system. This fabricated CL flow-through column has been successfully applied to determine nitrite in sausage samples with recoveries of 98-102%. These satisfactory results demonstrated that our studies pave a novel way toward flow-through column-based CRET using solid-state organic molecules as acceptors for signal amplification.

Effect of Internal Energy on the Repulsive Coulomb Barrier of Polyanions

The nature of the repulsive Coulomb barrier in isolated molecular polyanions is studied by means of the photodetachment dynamics of the S(1) excited state of the fluorescein dianion which is bound solely by the repulsive Coulomb barrier. Photoelectron spectra reveal a feature at a constant electron kinetic energy, regardless of the excitation energy. This is explained by using an adiabatic tunneling picture for electron loss through successive repulsive Coulomb barriers correlating to vibrationally excited states. This physical picture is supported by time-resolved photoelectron spectra, showing that the tunneling lifetime is also invariant with excitation energy.

Electronic spectroscopy of biomolecules in solution: fluorescein dianion in water

A combined and sequential Monte Carlo–quantum mechanics methodology is used to describe the electronic absorption spectrum of the fluorescein dianion in water. Different sets of 100 statistically relevant configurations composed of the solute and several solvent molecules are sampled from the Monte Carlo simulation for a posteriori quantum mechanical calculations of the spectra. In the largest case the configurations are composed of fluorescein and 90 explicit water molecules embedded in the electrostatic field of all remaining water molecules within a distance of 11.3 Å. These configurations include 305 atoms and 842 valence electrons, justifying the use of a semi-empirical approach. The electronic spectrum is then calculated using the INDO/CIS method. The solvatochromic shift of fluorescein in water, compared with in isolation, is calculated using the discrete and explicit solvent models. The use of electrostatically embedded explicit water molecules, in INDO/CIS calculations, gives a good description of the spectral shift of the fluorescein dianion in aqueous environment. The results are verified to converge both statistically and with respect to the number of explicit solvent molecules used.

Effect of Ionic Liquid on Prototropic and Solvatochromic Behavior of Fluorescein

The effect of a water-miscible room-temperature ionic liquid (IL), 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF(4)]), on the behavior of a common and popular probe fluorescein is assessed. Depending on solubilizing milieu and conditions, fluorescein may exist in one or more of its prototropic forms (cationic, neutral (zwitterionic, quinoid, and lactone), monoanionic (phenolate and carboxylate), and dianionic) in the solution. Information regarding the various prototropic forms of fluorescein in buffer-rich aqueous IL mixtures ([bmim][BF(4)] ≤ 50 wt %, mole fraction <0.1) at different pH is obtained via UV-vis absorbance and fluorescence. The addition of [bmim][BF(4)] is found to cause lactonization of fluorescein in the solution. Hydrolytic instability of IL [bmim][BF(4)] is observed to be much lower than that of NaBF(4). Absorbance and fluorescence from fluorescein are observed to be insignificant when dissolved in neat [bmim][BF(4)], suggesting the presence of predominantly the lactone form. The IL [bmim][BF(4)] behaves similar to apolar media in this context. It is demonstrated that various prototropic forms of fluorescein may be generated within [bmim][BF(4)] by the addition of buffer of appropriate pH. Significant bathochromic shift in absorbance and fluorescence band maxima of dianionic fluorescein as concentration of [bmim][BF(4)] is increased correlates well with the decrease in the hydrogen-bond-donating acidity of the medium. The lack of good linear correlation between the Stokes shift and the orientational polarizability suggests the possible presence of specific Coulombic interaction between the IL cation and the fluorescein dianion within the mixture. It is established that IL [bmim][BF(4)] has an interesting and unique effect on the prototropic behavior of fluorescein.

On the Intrinsic Photophysics of Fluorescein

Not so bright: Ion trap mass spectrometry is used in conjunction with optical excitation and detection to study the intrinsic spectroscopic properties of the fluorescein cation, monoanion, and dianion in the gas phase. The dianion, which is a fluorescence quantum yield standard in solution, does not fluoresce significantly in the absence of solvent (see picture) because electron detachment is the favored deactivation pathway in the gas phase.

Lotus-Root-like One-Dimensional Polymetallocages with Drastic Void Adaptability Constructed from 4,4′-Bis(1,2,4-triazol-1-ylmethyl)biphenyl and Zn(II) or Co(II) and Their Fluorescein Encapsulation Properties

The reaction of 4,4′-bis(1,2,4-triazol-1-ylmethyl)biphenyl (L) with Zn(NO3)2·6H2O or Co(NO3)2·6H2O in the presence of NH4PF6 in water−methanol or water−acetone produced both {[ZnL3](PF6)2(H2O)2}n (1) or {[CoL3](PF6)2(H2O)2}n (2). The metal ions in both complexes coordinate with six identical N atoms from six different ligands in an ideal octahedral environment where each ligand binds to two metal ions. Both complexes are one-dimensional triple-helical chains each containing an empty lotus-root-structured polymetallocage. The metallocage in 1 is fully empty, while the cage in 2 contains two water molecules. However, metallocages in 1 have a void volume of ∼200 A3, which is much smaller than the 1020 A3 void in 2. The void in 2 is comparable to the size of fluorescein. Both complexes are capable of encapsulating fluorescein anion in pH 9 aqueous solution to form {C20H10O5 ⊂ [ML3]}n, where C20H10O52− is fluorescein dianion, and M is Zn or Co. The encapsulation occurs via anion exchange. The cages in 1 are in...

Quantitative Imaging in the Laboratory: Fast Kinetics and Fluorescence Quenching

The introduction of imaging technologies into chemical and biochemical analysis has revolutionized fields as diverse as atomic spectroscopy and drug discovery. Traditionally, scientific cameras and associated analysis software have been expensive, difficult to use, and not widely distributed in teaching laboratories. Despite the widespread use of imaging technologies in many areas of chemistry, biology, biochemistry, biophysics, and medicine, they are not widely found in the undergraduate laboratory. This article describes a simple, inexpensive, experiment based on quantitative imaging. The experiment allows students to use quenching of fluorescein dianion to measure rate constants near 2 x 109 L mol-1 s. This experiment has been used during four academic years in an intermediate biochemistry course by ∼75 students. In this context, it was used to introduce students to the principles of fluorescence quenching, which is used in a variety of ways in modern biophysical chemistry. It may also be a suitable platform for lab modules for physical chemistry.

Quantum Mechanical Studies of Intensity in Electronic Spectra of Fluorescein Dianion and Monoanion Forms

Quantum chemical ab initio investigations with full geometrical optimizations of dianion and monoanion fluorescein molecules in the ground state were performed by applying the density functional theory (DFT) B3PW91/6-311G** model to clarify the difference in the geometrical and electronic structure of the dianion molecule and that of monoanion in influencing fluorescence. The mechanism of fluorescence of dianion and monoanion is explained and the reason for intensive fluorescence of dianion compared with that of monoanion is studied