Fluorescein In Aqueous Solution Research Articles

Fluorophore spectroscopy in aqueous glycerol solution: the interactions of glycerol with the fluorophore.

A common perception exists that glycerol provides an inert-like environment modifying viscosity and index of refraction by its various concentrations in aqueous solution. Said perception is herein challenged by investigating the effects of the glycerol environment on the spectroscopic properties of fluorescein, as a representative fluorophore, using steady-state and time-resolved techniques and computational chemistry. Results strongly suggest that the fluorescence quantum yield, measured fluorescence lifetime (FLT), natural lifetime and calculated fluorescence lifetime are all highly sensitive to the presence of glycerol. Glycerol was found to impact both the ground and first excited states of fluorescein, quenching and modifying both absorption and emission spectra, affecting the fundamental electrical dipoles of the ground and first excited singlet states, and lowering FLT and quantum yield. Furthermore, the Stern-Volmer, Lippert-Mataga, Perrin and Strickler-Berg relations indicate that glycerol acts upon fluorescein in aqueous solution as a quencher and alters the fluorescein geometry. Predictions made by computational chemistry impressively correspond to experimental results, both indicating changes in the properties of fluorescein at around 35% v/v aqueous glycerol, a clear indication that glycerol is not an innocent medium. This study proposes the Strickler-Berg relation as a means of detecting non-negligible effects of a hosting medium on its host fluorophore. These new insights on the molecular structures, the interactions between glycerol and its host fluorophore, and the effects of one on the other may be essential for understanding fundamental phenomena in chemistry and related fields.

Capturing the value in printed pharmaceuticals – A study of inkjet droplets released from a polymer matrix

The future of personalised combination dosages will rely on the programming and delivery of multiple, separate APIs, their carrier fluids and excipients to a stable matrix, where each will remain separate until it is needed to be released. A recent technique has demonstrated how to print, capture and release materials from a polymer matrix using inkjet printing, a low cost and customisable technique. Droplets of a formulation are delivered to a fluid polymer matrix, where they are imbibed and remain pinned just under the upper surface, held in place by a balance of interfacial energies. Once the surrounding matrix cures and solidifies, the coating or matrix has trapped the formulation, but each drop has a small opening or pore to the outside that will allow delivery through diffusion. However, while the trapping mechanism has been explored in detail, to-date the release involved in this delivery has never been studied or quantified to the same level. Here we show a first study to quantify the release of a model system from a polymer matrix. An aqueous fluorescein solution is delivered and trapped, with release demonstrated to an agarose gel and aqueous environments. The work reveals that the balance of interfacial tensions prevents a reliable release until low concentrations of surfactant are included. This provides a route forward to further explore stabilising combinations of drugs within one material using a digitally controlled and affordable technique.

Controlling Shapes in a Coaxial Flow Focusing Microfluidic Device: Experiments and Theory.

A coaxial flow focusing PDMS (polydimethylsiloxane) microfluidic device has been designed and manufactured by soft lithography in order to experimentally study a miscible inner flow. We studied a coaxially focused inner flow (formed by an aqueous fluorescein solution) which was fully isolated from all microchannel surfaces by an additional water outer flow. Different flow rates were used to produce a variety of flow ratios and a 3D reconstruction of the cross-section was performed using confocal microscope images. The results showed an elliptical section of the coaxially focused inner flow that changes in shape depending on the flow rate ratio applied. We have also developed a mathematical model that allows us to predict and control the geometry of the coaxially focused inner flow.

Polyvinyl pyrrolidone capped fluorescent anthracene nanoparticles for sensing fluorescein sodium in aqueous solution and analytical application for ophthalmic samples.

Based on the known complexation ability between polyvinyl pyrrolidone (PVP) and fluorescein sodium (FL Na(+)), fluorescent PVP capped anthracene nanoparticles (PVP-ANPs) were prepared using a reprecipitation method for detection of fluorescein in aqueous solution using the fluorescence resonance energy transfer (FRET) approach. A dynamic light scattering histogram of PVP-ANPs showed narrower particle size distribution and the average particle size was 15 nm. The aggregation-induced enhanced emission (AIEE) of PVP-ANPs was red shifted from its monomer by 1087.22 cm(-1). The maximum emission was seen to occur at 420 nm. The presence of FL Na(+) in the vicinity of PVP-ANPs quenched the fluorescence of PVP-ANPs because of its adsorption on the surface of PVP-ANPs in aqueous suspension. The FL Na(+) and PVP-ANPs were brought close enough, typically to 7.89 nm, which was less than the distance of 10 nm that is required between the energy donor-acceptor molecule for efficient FRET. The quenching results fit into the Stern-Volmer relationship even at temperatures greater than ambient temperatures. The thermodynamic parameters determined from FRET results helped to propose binding mechanisms involving hydrophobic and electrostatic molecular interaction. The fluorescence quenching results were used further to develop an analytical method for estimation of fluorescein sodium from ophthalmic samples available commercially in the market.

Effect of the glyceryl monooleate-based lyotropic phases on skin permeation using in vitro diffusion and skin imaging

Glyceryl monooleate (GMO) is a polar lipid that can exist in various liquid crystalline phases in the presence of different amounts of water. It is regarded as a permeation enhancer due to its amphiphilic property. Various phases of GMO/solvent system containing sodium fluorescein were prepared to compare permeability using confocal laser scanning microscopy (CLSM). GMO was melted in a vial in a water bath heated to 45 °C. Propylene glycol and hexanediol were homogeneously dissolved in the melted GMO. Sodium fluorescein in aqueous solution was diluted to various ratios and thoroughly mixed by an ultrasonic homogenizer. Each GMO/Solvent system with fluorescein was applied onto the epidermal side of excised pig skin and incubated overnight. CLSM was performed to observe how the GMO/solvent system in its different phases affect skin permeability. Cubic and lamellar phase formulations enhanced the fluorescein permeation through the stratum corneum. A solution system had the weakest permeability compared to the other two phases. Due to the amphiphilic nature of GMO, cubic and lamellar phases might reduce the barrier function of stratum corneum which was observed by CLSM as fluorescein accumulated in the dermis. Based on the results, the glyceryl monooleate lyotropic mixtures could be applied to enhance skin permeation in various topical and transdermal formulations.

Diffusion and Aggregation of Sodium Fluorescein in Aqueous Solutions

The diffusion and aggregation of sodium fluorescein in aqueous solutions was investigated adopting density functional theory (DFT) and molecular dynamics (MD) simulations. First, DFT calculations in implicit water were used to determine minimum energy structure and atomic charges of the solute, which were then used as input for explicit water MD simulations. The self-diffusion coefficient of sodium fluorescein was calculated using the Einstein equation, computing the mean square displacement from 24 ns trajectories. The calculated diffusion coefficient, 0.42 · 10(-5) cm(2) s(-1), is in good agreement with literature experimental data. The simulations confirmed the tendency of fluorescein to form dimers. In order to achieve a deeper understanding of aggregation phenomena, the dimer geometry was investigated through DFT calculations both in vacuo and in implicit water using different functionals and solvation theories. The results showed that dimerization does not occur in vacuo, as charge repulsion dominates, and that the minimum energy dimer structure is symmetric and stabilized by edge-to-face π-π interactions. The interaction energy was computed both at the DFT level and through MD simulations using Umbrella Sampling. The free interaction energy calculated with the WHAM and Umbrella Integration protocol, -1.3 kcal/mol, is in good agreement with experimental data, while the value determined using DFT calculations is significantly smaller and depends largely from the chosen functional and the computational methodology used to determine the solute-solvent boundary surface.

The influence of β-cyclodextrin on acid–base and tautomeric equilibrium of fluorescein dyes in aqueous solution

The protolytic equilibrium of fluorescein in aqueous solutions was studied in the presence of cycloheptaamylose (β-cyclodextrin, or β-CD). The constants of stepwise ionization of the dye ( H 3 R + ⇆ H 2 R ⇆ HR - ⇆ R 2 - ), K a0, K a1, and K a2 were determined using vis-spectroscopy at ionic strength 0.05 M (NaCl + buffer) and 25 °C. In the presence of 0.0086 M β-CD, the indices of ionization constants are as follows: p K a0 = 1.21 ± 0.12, p K a1 = 5.08 ± 0.03, p K a2 = 6.35 ± 0.02. The changes in these p K as, as compared with the values determined without cyclodextrin, are unequal. Namely, the p K a0 value decreases by 1.0, while the p K a1 value increases by 0.7. Thus, the introduction of β-CD allows to govern the ratios K a0/ K a1 and K a1/ K a2, which are equal to, respectively, 141 and 151 in water, and 7.4 × 10 3 and 18.6 with cyclodextrin added. Rationalization of the observed phenomenon is possible taking into account the detailed scheme of protolytic equilibrium. Conclusions concerning tautomerism of dye molecules were deduced from absorption spectra; the fractions of tautomers, tautomerization constants, and microscopic ionization constants were evaluated. These data allow concluding that the main reason for the aforementioned p K a alterations is the binding of H 2R by the cyclodextrin cavity accompanied by turning these neutral species into the colorless lactone. The host–guest interaction of neutral species of fluorescein isothiocyanate, 2,7-dichlorofluorescein, and 3′,4′,5′,6′-tetrachlorofluorescein also results in the cyclodextrin-assisted shift of tautomeric equilibrium. Such nature of interactions is proved by the addition of competing agents, camphor-4-carboxylic acid and sodium n-nonylsulfonate, which results in the removing of neutral dye species from the cycloheptaamylose cavity.

Measurement of Absorption and Scattering With an Integrating Sphere Detector: Application to Microalgae.

A spectrometer with an integrating sphere (IS) detector was used to measure the absorbance due to scattering and absorption. Analysis of the measurement process showed that two measurements of the absorbance, one with the cuvette placed in the normal spectrometer position, and the second with the cuvette placed next to the entrance aperture of the IS detector, provide enough information to separate the contributions from scattering and molecular absorption. Measurements were carried out with mixtures of microsphere and chromophore solutions. Two cases were examined: microspheres suspended in an aqueous fluorescein solution, and microspheres suspended in an aqueous holmium oxide solution. In both cases, the proposed measurement model gave results which were in good agreement with the expected response. Measurements on microalgae suspensions yielded a molecular absorption contribution and a scattering contribution. The scattering contribution had significant spectral structure which was inversely related to the molecular absorption contribution. The absorption and scattering contributions may provide independent information on the status of chlorophyll molecules and the structure of chloroplasts in microalgae.

Frequency-domain Measurement of the PhotodegradationProcess of Fluorescein¶

The frequency-domain technique is applied to measure the photodegradation rate of fluorescein in aqueous solutions. The illuminating light is modulated, and the changes in fluorescence from the illuminated region are detected synchronously. A constant flow rate is imposed on the fluorescein solution to control the mass transport of fluorescein into the illuminated region. The fluorescence response is described by a model that assumes that photodegradation occurs from the triplet excited state. The predictions of the model are consistent with the observed variations in the fluorescence response with flow rate, modulation frequency and incident power. We discuss in this article how the dependence of the model parameters on experimental conditions can be used to infer the photodegradation rate as well as some of the details of the photodegradation mechanism. The results are consistent with the known mechanism of photodegradation of fluorescein. The frequency-domain technique gives a photodegradation rate of 53 s−1 in an air-saturated solution and 37 s−1 in solutions purged with argon gas.

Time-Resolved Detection of Hot Electron-Induced Electrochemiluminescence of Fluorescein in Aqueous Solution

Strong electrogenerated chemiluminescence (ECL) of fluorescein is generated during cathodic pulse polarization of oxide-covered aluminum electrodes and the resulting decay of emission is so sluggish that time-resolved detection of fluorescein is feasible. The present ECL in aqueous solution is based on the tunnel emission of hot electrons into the aqueous electrolyte solution, which probably results in the generation of hydrated electrons and hydroxyl radicals acting as redox mediators. The successive one-electron redox steps with the primary radicals result in fluorescein in its lowest excited singlet state. The method allows the detection of fluorescein (or its derivatives containing usable linking groups to biomolecules) over several orders of magnitude of concentration with detection limits well below nanomolar concentration level. The detection limits can still be lowered, e.g., by addition of azide or bromide ions as coreactants. The results suggest that the derivatives of fluorescein, such as fluorescein isothiocyanate (FITC), can be detected by time-resolved measurements and thus be efficiently used as electrochemiluminescent labels in bioaffinity assays.

Hot electron-induced electrochemiluminescence of fluorescein in aqueous solution

Strong electrogenerated chemiluminescence (ECL) of fluorescein is generated in aqueous solution during cathodic pulse polarization of oxide-covered aluminum electrodes. The present method is based on the injection of hot electrons into the aqueous electrolyte solution, which probably results in the generation of hydrated electrons as reducing mediators. The successive one-electron redox reactions result in fluorescein in its lowest excited singlet state, i.e., the emission spectrum is similar to the corresponding fluorescence emission spectrum. This ECL can be enhanced by added coreactants, e.g., by peroxodisulfate, peroxodiphosphate, hydrogen peroxide and azide ions. The method can detect fluorescein over several orders of magnitude of concentration with detection limit below nanomolar concentration level in the presence of azide ions. The results suggest that the derivatives of fluorescein, such as fluorescein isothiocyanate, can be used as electrochemiluminescent labels in aqueous solution in bioaffinity assays carried out on the surface of thin aluminum oxide film-coated aluminum electrodes. This cathodic ECL is mainly based on the one-electron oxidation of fluorescein by the cathodically produced oxidizing radicals followed by reduction of the formed semioxidized fluorescein by hot or hydrated electrons back to the original oxidation state which finally emits light.

Fabrication of single nanofluidic channels in poly(methylmethacrylate) films via focused-ion beam milling for use as molecular gates

Focused-ion beam (FIB) milling provides rapid fabrication of individual cylindrical submicrometer channels with reproducible dimensions (±5% diameters) through 8-μm thick poly(methylmethacrylate) (PMMA) films. PMMA films are spincast on sacrificial Si carriers and sputter-coated with Au before the 30-kV gallium FIB milling process. By adding a trace amount of poly(ethyleneoxide) and poly(dimethylsiloxane) to the PMMA solution before casting, the films can be released for subsequent mounting in microfluidic devices to create hybrid microfluidic-nanofluidic multilevel architectures. In situ FIB sectioning demonstrates the smooth cylindrical surface within the pore. Placing a milled film in contact with an aqueous fluorescein solution fills the channel by capillary action, as verified by confocal fluorescence microscopy. Confocal fluorescence of dyed films reveals that the pores span the thickness of the PMMA film. Small arrays of channels with a defined number and density and arbitrary in-plane spatial arrangement are fabricated with this process, allowing a unique testbed for high aspect ratio nanofluidic devices.

Photodegradation of Fluorescein in Solutions Containing n-Propyl Gallate

The frequency domain technique was applied to measure the effect of n-propyl gallate (nPG) on the apparent photodegradation rate of fluorescein in aqueous solutions. The illuminating light was modulated and the change in fluorescence from the illuminated region was detected synchronously. A constant flow rate was imposed on the fluorescein solution to control the mass transport of fluorescein into the illuminated region. The photodegradation response was described by a model which assumed two steps: (1) singlet oxygen production via energy transfer between the excited triplet state of fluorescein and molecular oxygen in the ground triplet state and (2) photodegradation via the interaction of fluorescein with singlet oxygen. It was assumed that nPG affects the photodegradation of fluorescein by quenching the oxygen singlet state. In the context of this model, the rate of singlet oxygen quenching by nPG was found to be (1.3 ± 0.2) × 109 s-1M-1. The product of the singlet oxygen photosensitization rate, kox...

Frequency-domain measurement of the photodegradation process of fluorescein.

The frequency-domain technique is applied to measure the photodegradation rate of fluorescein in aqueous solutions. The illuminating light is modulated, and the changes in fluorescence from the illuminated region are detected synchronously. A constant flow rate is imposed on the fluorescein solution to control the mass transport of fluorescein into the illuminated region. The fluorescence response is described by a model that assumes that photodegradation occurs from the triplet excited state. The predictions of the model are consistent with the observed variations in the fluorescence response with flow rate, modulation frequency and incident power. We discuss in this article how the dependence of the model parameters on experimental conditions can be used to infer the photodegradation rate as well as some of the details of the photodegradation mechanism. The results are consistent with the known mechanism of photodegradation of fluorescein. The frequency-domain technique gives a photodegradation rate of 53 s(-1) in an air-saturated solution and 37 s(-1) in solutions purged with argon gas.

Raman and FTIR spectroscopies of fluorescein in solutions

Raman and Fourier transform-infra red (FT-IR) spectroscopies of fluorescein in aqueous solutions have been investigated in the pH range from 9.1 to 5.4. At pH 9.1 fluorescein is in the dianion form. At pH 5.4, fluorescein is a mixture of monoanion (∼85%), dianion and neutral forms (together ∼15%). The fluorescence quantum yield drops from 0.93 for the dianion form to 0.37 for the monoanion form. The Raman and FT-IR studies focused on the frequency range from 1000 to 1800 cm −1 which contains the skeletal vibrational modes of the xanthene moiety of fluorescein. At pH 9.1, the spectroscopic feature of fluorescein dianion are consistent with a picture of an electron delocalized among the xanthene moiety and two identical oxygens attached to opposite ends of the xanthene moiety, forming a very symmetric structure. The characteristic of fluorescein dianion is the presence of the phenoxide-like stretch at 1310 cm −1. At pH 5.4, fluorescein monoanion has lost the symmetric structure characteristic of the dianion. The spectra of the monoanion have distinctive contributions from the phenolic bend at 1184 cm −1. The assignments of the vibrational bands shown in Raman and FT-IR spectra are given based on both literature and the ab initio calculations at the Hartree–Fock level with HF/6-31++G* basis set. Excellent correlation is found between the experimental and calculated spectra.

Novel Experimental Technique for the Absolute Determination of Quantum Yields

A new method for the absolute determination of the quantum yield based on the trapping of fluorescence light within a cylinder is discussed. First the trapping probability of photons—which are emitted in transparent rods or fluid-filled tubes—is investigated theoretically. The studies are based on the extension of light governed by the laws of reflection and refraction. The effect of nonhomogeneous distribution of the centers of fluorescence light emission on the calculation procedures is taken into account. Then the adaptation of the theoretical results to a new practical method is shown. The quantum yield as a function of the exciting wavelength is determined in one experimental cycle. The discussion of the method is performed by measurements on fluorescein in aqueous solution. The results are compared to literature data. The conformity demonstrates the applicability of the new method.

Spectral properties of the prototropic forms of fluorescein in aqueous solution

The commonly used fluorescent probe, fluorescein, can exist in seven prototropic forms. We have used global analysis procedures to reanalyze the absorption data of Diehl and Horchak-Morris (Talanta 34, 739-741, 1987) in terms of five alternative ionization models. We identify the forms of fluorescein present in aqueous solution and the pK a of each ionisation transition. The pKa values of the neutral xanthene, carboxylic acid, and cationic xanthene groups are 6.3, 3.1-3.4, and 3.1-3.4, respectively, and the pKa value of lactonization is 2.4. As a consequence, the neutral form of fluorescein is a mixture of the lactone (70%), zwitterionic (15%), and quinoid (15%) forms. A knowledge of the forms present in solution permits the characterization of their spectral properties. It is shown that the quinoid and monoanion forms have similar absorption spectra, while the zwitterion spectrum is similar to that of the cation but blue-shifted by 3 nm. The emission spectra of the monoanion and quinoid forms are also identified and shown to be similar but not identical. A model for the excited-state reactions of fluorescein is presented.

Photochemical hydrogen evolution from alkaline solutions of xanthene dyes

The effect of the composition of five xanthene dyes (photoabsorber and photosensitizer) on the hydrogen photoproduction from aqueous solutions was investigated. It is suggested that triethanolamine (TEOA) can quench the excited singlet state of fluorescein in aqueous solution (1 × 10 −4 mol l −1, pH 12.5). As a result, a reduced form of fluorescein is produced, which can further reduce H + to H 2 in the presence of K 2PtCl 6 (5 × 10 −5 mol l −1). The quantum yield of H 2 photoproduction ( Φ H2) is 0.024. For tetraiodofluorescein (erythrosin) in aqueous solution (1 × 10 −4 mol l −1), pH 12.5; K 2PtCl 6, 5 × 10 −5 mol l −1), Φ H2 is as high as 0.30 owing to the inner molecular heavy atom effect. The quantum yields of fluorescence and intersystem crossing, Φ F and Φ ISC, were measured and compared with Φ H2 for all the investigated dyes. The results show an important ability of xanthene dyes to enhance hydrogen photoproduction from alkaline aqueous solutions.

Determination of equilibrium constants by chemometric analysis of spectroscopic data

We describe a chemometric method to determine equilibrium constants with high accuracy from spectroscopic titrations. Knowledge of component spectra is not required for the analysis, and titrations can be analyzed even when the titration end points are not reached. In fact, the component spectra are determined in the analysis. The analysis is based on a decomposition of the recorded spectra into a product of target and projection matrices using NIPALS. The matrices are then rotated to give the correct concentrations and spectral profiles of the components utilizing the functional form of the equilibrium expression. The output of the analysis is the equilibrium constant and the component spectral profiles. The analysis is highly accurate and can be performed on a standard personal computer. As examples, we determine the protolytic constant for the equilibrium between the anionic and dianionic forms of fluorescein in aqueous solution and the dimerization constant of benzoic acid in n-heptane.

PH and excitation wavelength dependence of the thermal lens signal of fluorescein in aqueous solutions

It is shown that the thermal lens signal of fluorescein in aqueous solutions depends on the molecular form of the solute even after correction for fluorescence. Possible factors that may affect the thermal lens response are photochemical reactions and vibration-to-translation energy transfer from the solute to the solvent. These are considered in relation to the existence of excited-state proton transfer reactions.