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Biomarkers are essential part of daily medical practice. Currently, biomarkers are being used both for diagnostic and prognostic purposes. There are many approaches e.g. ELISA by which biomarker levels are detected from patient samples. However, all these approaches are laborious, time consuming and expensive. There is therefore a general need for exploring new technique which can overcome these drawbacks. Here, we present a preliminary study for detection of serum biomarkers by fluorescence correlation spectroscopy (FCS) based diagnostic technique. FCS is a technique basically used for spatial and temporal analysis of molecular interactions of extremely low-concentration biomolecules in solution. FCS is able to measure diffusion time of the fluorescent molecules passing through the open detection volume and it can also measure the average number of fluorescent molecules passing through the detection volume. Because diffusion speed is correlated with shape and molecular mass of the fluorescent molecule, this property makes it possible to study the complex formation between a small fluorescently labelled and a large unlabelled molecule. In this preliminary study, we utilize this FCS property for detection of serum biomarker. Further studies on various pathological serum samples are warranted to explore further aspects of this technique.

In a comprehensive picture of inclusion com- plex formation of the highly fluorescent dye coumarin-6 (C6) and betacyclodextrin (beta-CD), which was obtained using various fluorescence spectroscopic methods, it was demonstrated that up to three beta-CD rings can thread on the rod like dye molecule. Interaction of coumarins and modified coumarins with cellular organelles or proteins has been reported in several publications. Especially 7-amino- coumarins are characterized by unique properties like high fluorescence quantum yield and are thus already used successfully in different areas, like staining of fluorescent nanoparticles. We could show that Coumarin-6 made sol- uble by complexation with beta-cyclodextrin is able to stain eukaryotic cells specifically dependent on their origin and cellular behaviour. The staining reaction is indepen- dent from pH, is photo stable, and shows no cross talk with proteins in the cytoplasm and other staining procedures or erythrocytes. Staining with coumarin 6/cyclodextrin com- plexes can thus be used for fast discrimination of different cell types. Importantly, it could be shown that the ideal staining reaction is dependent on the stoichiometry of the complex-formation.

Fluorescence correlation spectroscopy (FCS) allows determining diffusion and relaxation properties of fluorescent molecules. It requires only extremely small amounts of sample, down to picomolar concentrations, in an effective analysis volume of a few femtoliters. In essence, FCS determines the autocorrelation of fluorescence fluctuations caused by single labeled molecules passing through the confocal volume of a microscope equipped with a suitable detector; it permits investigating interactions of (macro)molecules, even in single cells. We present an FCS protocol for exploring, under in vitro conditions, the dynamic processes that take place during the early steps of virus infection. We cover two important issues of rhinovirus research, the kinetics of directional RNA release, and virus-receptor interactions exemplified by using human rhinovirus type A2 (HRV-A2) as a model.

Supramolecular host–guest complexes formed between dye molecules, which are absorbing and fluorescing in the visible range, and cyclodextrin molecules are studied with the aim to generate a self-assembled nanostructured layer on a surface. Studies in solution show that an aminocoumarin dye, i.e. coumarin-6, binds strongly to β-cyclodextrin and yields fluorescent structures. The complex formation is substantiated by 17 nm blue shift of the fluorescence spectrum. The binding of thiolated cyclodextrin, 6-monodeoxy-6-monothio-β-cyclodextrin, to gold surface is then studied by induced change in the second-harmonic signal intensity generated from the surface. The saturation times of surface adsorption, as a measure of binding kinetics, are estimated for thiolated cyclodextrin as well as hexadecanethiol. The results support formation of self-assembled layers of the host molecules, which can serve as basis for the formation of supramolecular host–guest surface layers.

The excited states of various di-alkyl-aminobenzonitriles (ABN) emitting dual fluorescence are investigated theoretically by a combination of density functional theory and multireference configuration interaction (DFT/MRCI). Experimentally, it was found that intramolecular charge transfer (ICT) emission increases on expense of the normal fluorescence with increasing n-alkyl chain length. The long wavelength ICT band becomes the predominant fluorescence band in diisopropylaminobenzonitrile (DIABN) even in an apolar environment. The calculated energy of both the planar and twisted ICT (PICT and TICT) state increases slightly with increasing chain length relative to the energy of the first excited state at optimized planar ground-state geometry. The good agreement of the calculated TICT state dipole moment with the experimental ICT state dipole moment leads us to the conclusion that the dual fluorescence of all linear substituted di-alkyl ABNs originate from a TICT state. The bulky system DIABN is twisted by 33° already in the ground state and complete decoupling of donor and acceptor units takes place without a significant energy barrier. In contrast, planarization toward the PICT state is energetically disfavored. Thus, the red-shifted fluorescence of DIABN is also assigned to emission out of the TICT state. © 2001 John Wiley & Sons, Inc. Int J Quantum Chem 84: 149–156, 2001

The structures, spectroscopic and photophysical properties of the inclusion complexes between 4-OH-biphenyl and cyclodextrins (CD) in water were studied theoretically and experimentally. The complex structures were predicted using a dynamic Monte Carlo procedure including solvation effects and analyzed via computed and experimentally determined circular dichroism. The interpretation of the induced circular dichroism spectra indicated the formation of stable 2:2 complexes between the probe and alpha-CD, while 1:1 structures account for the circular dichroism induced by beta- and gamma-CD. Fluorescence emission quantum yields and lifetimes, measured as a function of the CD concentration, confirmed the formation of this higher order complex with alpha-CD. Prototropic equilibration in the singlet excited state was found to be depressed in 2:2 complexes due to the hydrophobic environment of the OH groups, while it remained unperturbed in 1:1 complexes, where the substituent is exposed to the aqueous environment. Triplet-triplet absorption and triplet quenching data supported this interpretation. The photophysical properties of both the 1:1 and the 2:2 complexes are characterized by a significant reduction of the nonradiative decay rates.

Fluorescence properties of 1-heptanoylpyrene in homogeneous solutions and incorporated into liposome membranes have been measured. The fluorescence intensity of the alkanoylpyrene is small in neat hydrocarbon solutions (Qf = 0.006) but increases with increasing solvent polarity. On the contrary, Qf of the 1-alkylpyrene is larger by a factor of 10 in a hydrocarbon solvent and decreases slightly in polar solvents. In alcohols the fluorescence yield of 1-heptanoylpyrene becomes considerably larger in comparison to a nonhydroxylic environment and nearly identical to that of heptylpyrene in the same solvent. The fluorescence spectra shift bathochromically in that polar environment. The fluorescence properties of 1-heptanoylpyrene incorporated in a liposome membrane indicate that the probe molecules are well embedded within the hydrophobic core of the bilayer. Semiempirical calculations were performed for 1-ethanoylpyrene and 1-ethylpyrene as model compounds in order to analyze the influence of solvent polarity and of hydrogen bonding on excited state energies and fluorescence properties of 1-acyl derivatives of pyrene. Hydrogen-bonding increases the energy gap between the lowest singlets, which are nearly degenerated, and the 3π*-n triplet and reduced consequently the intersystem crossing efficiency. But also a change in the sequence of the excited 1π*-π states is concluded from experimental data and should contribute to the increase of the fluorescence yield upon hydrogen bonding.

The efficient fluorescence probe 6-propanoyl-2-(N,N-dimethylamino)naphthalene (PRODAN) shows a significant Stokes shift in polar solvents. Neither experimental nor theoretical studies performed up to now yield a definitive description of the character of the emissive state. Results of the recently developed DFT/SCI method are compared to semiempirical calculations with the spectroscopic ZINDO/S parametrization and the AM1/CISD Hamiltonian which was not parametrized for computation of spectroscopic properties. The most reliable results are obtained with the DFT/SCI and AM1/CISD approaches. The absorption spectrum of PRODAN is excellently reproduced by the DFT/SCI calculations, and the AM1/CISD results are superior to ZINDO/S. These results allow the assignment of the red-shifted fluorescence band to both emission of a highly polar dimethylamino twisted intramolecular charge transfer state (N-TICT) and a geometrically unchanged planar intramolecular charge transfer (PICT) state. A comparison with 2-(N,N-dim...

The photophysical properties of 10-decyl- and 10-propenyl-9-acridones, important emitters in electro- and chemiluminescence, are investigated in dependence of the solvent polarity and its hydrogen bonding ability at room temperature as well as in frozen matrix at 77 K. Fluorescence is inefficient in non-polar solvents at room temperature due to fast intersystem crossing but increases with solvent polarity due to the inversion of the S 1 (ππ ∗ ) and the T (nπ ∗ ) states. In the case of 9-[10-(1-propenyl)]-acridone the dependencies of the fluorescence spectrum and quantum yield on the ET(30) solvent parameters indicate that intermolecular hydrogen bonded solute-solvent complexes are formed in the excited state, inducing efficient non-radiative deactivation.

The photophysics of 9,9‘-bianthryl (BA) were investigated by means of fluorescence spectroscopy, nanosecond transient-absorption spectroscopy, and semiempirical calculations. Fluorescence spectra and lifetimes were measured in more than 50 solvents in order to get a detailed picture of the solvent dependence. The results show that the fluorescence lifetime is constant in solvents of low polarity (D 5), thus providing direct proof of the charge-separated character of the fluorescent ...

Contbrmational energies of PRODAN (6-Propionyl-2-(dimethylamino)-naphthalene) have been calculated by Hartree-Fock ab initio methods as a function of the twisting and wagging motion of the amino and the rotational motion of the propionyl group. In agreement with experimental data, PRODAN is found planar in its optimum geometry. The CIS method is used to compute molecular orbital energies, excitation energies and dipole moments in dependence of the rotational isomerization of the dimethylamino group. The calculations allow the peculiar fluorescence properties of PRODAN to be explained by the TICT model.

The fluorescence properties of several pyrazoloquinoline derivatives were performed by stationary as well as by time-resolved spectrosopy. Non-donor-substituted compounds show a high quantum yield; transition dipole moments of absorption and fluorescence as well as experimental and calculated lifetimes are in excellent agreement. The donor-substituted compound DMA-DPPQ exhibits a charge transfer fluorescence in polar solvents. Additionally, dual fluorescence appears in polar protic solvents. The nature of the charge transfer state is discussed with respect to the the TICT model.

A new push-pull stilbene was synthesized, and its photophysical properties investigated by spectroscopic and theoretical methods. Solvatochromic shifts in the fluorescence were in accordance with Onsager's continuum model, whereas shifts in the absorption spectra only correlated with the polarizability of the solvents. Excited state dipole moments were determined and compared to semi-empirically calculated values. With increasing solvent polarity the fluorescence quantum yield increases steeply as the solvent dielectric permittivity exceeds 8. It is concluded that a strongly luminescent and highly dipolar π, π ∗ state becomes the lowest excited state in polar solvents, and radiative deactivation therefore competes effectively with non-radiative pathways. Preventing internal rotation of molecular subunits is thus no prerequisite for obtaining highly fluorescent push-pull stilbenes.

In addition to the yellow emission ascribed to the J aggregates of 1,1′-diethyl-2,2′-cyanine, this compound also exhibits a high-energy emission around 400 nm in dilute aqueous solutions (c

Steady-state and dynamic photophysical characteristics of a number of 4- and 5-amino-2-phenylindan-1,3-dione derivatives have been studied at 293 and 77 K in solvents of widely different polatiry and hydrogen-bonding ability. The observed changes of the fluorescence Franck-Condon transition energy, and of the fluorescence quantum yields and the singlet lifetimes, as well as the solvent deuterium-isotope effect, indicate the formation of intermolecular hydrogen bonds in the excited state with protic solvents for the 5-amino compounds at room temperature.

The appearance of wavelength dependent photochemistry (H-abstraction) and fluorescence in some typical polar aromatics (i.e. phenols, anilines, indoles) is correlated to the energetic position of Rydberg type excited states. This behaviour is also demonstrated for carbazole as an example of a larger aromatic molecular system. In bichromophoric molecules, interrelation between excited states of valence and Rydberg type is demonstrated. The significance of higher excited state photochemistry for primary radiation chemical processes is discussed.

Nanosized materials are increasingly used in medicine and biotechnology but originate also from various aerosol sources. A detailed understanding of their interaction with cells is a prerequisite for specific applications and appraisal of hazardous effects. Fluorescence fluctuation methods are applied to follow the time-course of the translocation and distribution of fluorescent 20 nm polystyrene nanoparticles with negative surface charges in HeLa cells under almost physiological conditions. The experimental results demonstrate that singular particles enter the cell without significant contribution by endocytotic mechanisms and are distributed within the cytoplasm. Subsequently aggregation is observed, which can be blocked by cytotoxins, like Genistein and Cytochalasin B, interfering with cellular uptake processes. The observed non-active uptake is due to non-specific interactions with the cell surface and could be responsible for distribution of nanometer-sized materials in tissue.

The effects of the excitation wavelength on the quantum yields Q(eaq−) and QF of electron ejection and fluorescence respectively were measured for 4-cyano-N,N-dimethylaniline (CDMA) and related compounds in aqueous solutions. In the case of CDMA no electron ejection was found for excitation of the long-wavelength (1La, 1Lb) band system. At shorter wavelengths (λexc

Fluorescence spectra, quantum yields and lifetimes of p-cyano-N,N-dimethylaniline (CDMA) were studied in various binary mixtures of n -hexane with chlorinated hydrocarbons. Solvent effects on the equilibrium between normal emission and fluorescence originating from the highly dipolar twisted internal charge transfer (TICT) state arise predominantly from non-specific solute-solvent interactions. The non-radiative deactivation rate of the TICT state is affected primarily by the solvent composition and slows down with the stabilization of the TICT state in environments of high polarity.

The effect of addition of the cyclodextrins (CDx) α-CDx and β-CDx on the photophysics and photochemistry of the dimethoxybenzenes (DMB) 1,4-DMB and 1,2-DMB in aqueous solution has been investigated by means of absorption, circular dichroism, fluorescence, and nanosecond transient absorption spectroscopies. The experimental results are discussed in the light of model calculations on the structure and the circular dichroism spectra of the DMB−CDx complexes. The association of 1,2-DMB with both CDxs is much weaker than that of 1,4-DMB owing to steric hindrance. With β-CDx, 1,4-DMB forms 1:1 complexes with a high association constant (K = 630 M-1) in spite of incomplete inclusion. Complexation of 1,4-DMB with α-CDx strongly affects the photophysical properties of the guest: fluorescence is enhanced and triplet state decay slowed by a factor of 10 or more, depending on the decay reaction mode. Fluorescence enhancement is mainly due to a reduction of the rate constant for internal conversion. The effect of com...

The influence of temperature and of various solvents on fluorescence and electron ejection has been studied using monophenylphosphate. Heavy atom quenching experiments with Cs+ ions and the temperature dependence observed for the system show that electrons are not only emitted from the thermalized singlet state of aqueous monophenylphosphate (Q= 0.02 for T= 30°C and pH = 8.2) but probably also during relaxation prior to fluorescence (Q= 0.008). The significant decrease in fluorescence at higher excitation energies can be satisfactorily explained by an increase in photodetachment of e–aq or H according to the pH of the solution (reaching 0.23 at pH 8.2). From the results it was concluded that electron ejection occurs via an intermediate state which has very similar properties to c.t.t.s. states observed in the spectroscopy of inorganic anions.

The dependence of the fluorescence quantum yield, QF, on the energy of the exciting light was examined for a number of aromatic amines (aniline, N,N-dimethylaniline, 2-phenylethylamine, etc.) in various polar and non-polar solvents down to 200 nm. QF of aniline is constant throughout the first absorption system but decreases when the excitation reaches the second band. This effect is most pronounced in hydrocarbon solutions but small in polar solvents capable of forming hydrogen bonds, with the only exception being water. For N,N-dimethylaniline a wavelength dependence of QF is observed only in aqueous solutions. Concomitant to the QF decrease the H2 evolution in hydrocarbons and electron ejection in water increase. These effects are also observed for solutions of 2-phenylethylamine and are explained by the formation of weak intramolecular complexes. A further pathway, characteristic of the benzoic ring, is assumed to be responsible for the wavelength dependence of protonated anilines. The derived deactivation pattern is of general validity for simple polar aromatics.

The dependence of the fluorescence quantum yield Q(F) on the excitation wavelength (λexc) was studied for phenol, anisole, p-cresol, p-methylanisole, 3,5-dimethylphenol and 2,4,6-trimethylphenol in polar and apolar solvents. All the systems investigated, with the exception of anisole and p-methylanisole exhibit a marked decrease in Q(F) at lower λexc values in hydrocarbon solvents. Further it was established that in this case enhanced photochemical cleavage of the OH bond is mainly responsible for this effect. Owing to the formation of H-bonds this dependence diminishes in polar, strongly associating solvents like alcohols and ethers, but not in water.