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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.

The photophysical properties of indoline (I) and three of its derivatives, namely, N-methylindoline (MI), 5-cyanoindoline (CI), and 5-cyano-N-methylindoline (CMI), are studied in H-donating solvents of varying polarity. Based on measurements of fluorescence yield and lifetime, and of triplet yield and hydrated-electron formation, two distinct mechanisms of solvent-induced fluorescence quenching are evidenced. The first mechanism involves the cyano substituent and leads to an increase in the rate constant of internal conversion of one order of magnitude in ethanolic solution and of more than two orders of magnitude in water, as compared to solutions in n-hexane or acetonitrile. A similar trend had previously been observed in the case of 4-N,N-dimethylaminobenzonitrile (DMABN). The second mechanism reduces the fluorescence lifetimes of the non-cyanated derivatives in aqueous solution by one order of magnitude and is related to the formation of hydrated electrons. Neither of these mechanisms is influenced by methylation at the ring nitrogen. Quantum chemical calculations are performed on the ground and excited states of the hydrogen-bonded complexes between protic solvents and MI as well as CMI. Stable hydrogen-bonded configurations involving the CN substituent and a solvent OH group are found; these configurations are stable both in the ground and the first excited singlet states, whereas the corresponding complex at the ring amino nitrogen is stable in the ground state only. The CN--HO configuration is therefore a prime candidate for a mechanistic explanation of the observed quenching by the first mechanism. These findings may have useful applications for the design of fluorescence probes for water in biological systems.

There are proteins that are built of two structural domains and are deposited full-length in amyloid plaques formed in various diseases. In spite of the known differences in the mechanisms of folding of single- and multidomain proteins, no published studies can be found that address the role of the domain-domain interactions during misfolding and amyloid formation. By the discovery of the role of domain-domain interactions, here we provide important insight in the submolecular mechanism of amyloid formation. A model system based on yeast phosphoglycerate kinase was designed. This system includes the wild-type yeast phosphoglycerate kinase and single-tryptophan mutants of the individual N and C terminal domains and the complete protein. Electron microscopic measurements proved that amyloid fibrils grow from all mutants under identical conditions as for the wild-type protein. Misfolding and amyloid formation was followed in stopped-flow and manual mixing experiments on the 1 ms to 4 days timescale. Tryptophan fluorescence was used for selective detection of conformational changes accompanying the formation of the amyloidogenic intermediates and the growth of amyloid fibrils. The interactions between the polypeptide chains of the two domains direct the misfolding process from the early steps to the amyloid formation, and influence the final structure. The kinetics of misfolding is different for the individual domains, pointing to the significance of the amino acid sequence. Misfolding of the domains within the complete protein is synchronized indicating that domain-domain interactions direct the misfolding and amyloid formation mechanism.

Fluorescence correlation spectroscopy (FCS) was used to study the diffusion behavior of the glucocorticoid dexamethasone on the cell membrane of the mouse pituitary cell line AtT-20 to investigate the putative membrane glucocorticoid receptor (mGR). This putative membrane receptor is thought to mediate glucocorticoid effects independent from protein synthesis and the well-known intracellular glucocorticoid receptor (iGR), the so-called “nongenomic” effects. The high spatial resolution of the FCS technique allowed for focusing exclusively on the cell membrane; measurements were taken on living cells in solution after incubation with 60 nM fluoresceindexamethasone for 1 h without manipulation of the cells. FCS measurements were performed by moving the position of the laser focus in 1 μm steps through a single cell. In a scan through the cell a component typical for a receptor-bound ligand (diffusion constant 3 x 10-10 cm2 sec-1) becomes apparent in the autocorrelation function (ACF) at the position of the cell membrane whereas another component (diffusion constant 2 x 10-8 cm2 sec-1) typical for a ligand diffusing in the cytosol becomes predominant in the ACF curves inside the cell. After pre-incubation with unlabeled dexamethasone in 1000-fold excess the “receptor-typical” component on the cell membrane was absent. This result is the first direct evidence for the existence of a mGR on a living cell.

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.

Potential photosynthetic models, porphyrin dyads with various acceptors, that are separated by a diazaphenylene spacer, are synthesized by a new strategy. The amidine and vinamidinium salts are coupled by pyrimidine rings. In one example (n=1 in the figure), excitation of a flavin acceptor results in energy transfer to the porphyrin singlet (≈60 %) and population of a charge-separated state (≈40 %), which relaxes to the ground state on a subnanosecond time scale.

4-(4′-N,N-dimethylaminophenyl)-3,5-dimethyl-1,7-diphenyl-bis-pyrazolo-[3,4-b;4′,3′e]-pyridine (DMA-DMPP), was recently synthesized as an example of a bulky electron donor-acceptor compound and was examined by spectroscopic methods. The molecule consists of several subunits of different redox properties and the observed behavior was thus interpreted by an intramolecular charge transfer model. This hypothesis is validated by semiempirical excited state calculations. They are focused on the coupling of intramolecular charge transfer to various relaxation modes, e.g. rotation about single bonds leading to decoupling π systems. Rotation about the single bond connecting the dimethylanilino and heteroaromatic subunit results as the most important relaxation coordinate. The two moieties non orthogonal in the ground state become perpendicularly oriented in the optimum excited state. In this conformation the dipole moment also becomes maximal. These results agree satisfactorily with the observation of a marked red shift of its fluorescence in polar solvents and dual fluorescence found in polar, protic environment.

— The two main primary photoprocesses (electron ejection and H-atom release) for indole, 5-methoxyindole and N-methylindole in various polar and nonpolar solvents were studied as a function of the excitation energy and were correlated with the corresponding fluorescence quantum yields. In hydrocarbon solvents, N–H bond cleavage is the main primary photoprocess from the 1Bb band of the substrates with the exception of N-methylindole. In alcohols, both processes are of negligible importance. Hydrated electrons (e−aq) are ejected from the relaxed singlet states of all three compounds in aqueous solutions with a similar yield for excitation at 280 and 254 nm (1La and 1Lb states). The yield increases when the excitation is into the 1Bb band. The quantum yields of the two primary processes from the higher excited states are generally lower than the fraction of molecules not converting to the fluorescent state. This is explained by an efficient back reaction in competition with a thermally activated radical release from an intermediate state or radical pair formed from the S2 (1Bb) state. The non-occurrence of a photoionization energy threshold is discussed.