An insight into pH-induced changes in FAD conformational structure by means of time-resolved fluorescence and circular dichroism.

Optical properties of flavin adenine dinucleotide (FAD) moiety are widely used nowadays for biotechnological applications. Given the fundamental role played by FAD, additional structural information about this enzymatic cofactor can be extremely useful in order to obtain a greater insight into its functional role in proteins. For this purpose, we have investigated FAD behaviour in aqueous solutions at different pH values by a novel approach based on the combined use of time-resolved fluorescence and circular dichroism spectroscopies. The results showed that pH strongly affects time-resolved fluorescence emission and the analysis allowed us to detect a three-component decay for FAD in aqueous solution with pH-depending lifetimes and relative amplitudes. Circular dichroism data were analyzed by a multi-Gaussian fitting procedure and the trends of properly chosen parameters confirmed pH-depending changes. The comparison between the results obtained by these two optical techniques allowed us to improve the significance of the outcome of circular dichroism. This combined approach may provide a useful tool for biotechnological investigation.

Depth profiles in confocal optical microscopy: a simulation approach based on the second Rayleigh-Sommerfeld diffraction integral.

A method is presented for recovering the intensity depth profile, by confocal optical microscopy, in transparent and amorphous samples with low scattering. The response function of a confocal Raman microscope has been determined by using the second Rayleigh-Sommerfeld diffraction integral and scalar wave optics within paraxial approximation, taking into account the refractive index mismatch between the sample and the medium surrounding the objective lens. An iterative multi-fitting-scheme, based on the conjugate gradient method and Brent algorithm, allowed to fit several depth profile curves simultaneously and retrieve the beam waist, the signal amplitude and the position of the sample surface. The reliability and accuracy of the theoretical procedure has been investigated through comparison with experimental measurements of the Raman depth profiles for different pinhole diameters. The model is shown to provide accurate description of the effect of the mismatch of the refractive index and of the dependence of the Raman signal on the depth with discrepancies lower than 3%. This procedure constitutes a first step towards the development of a manageable theoretical framework, amenable to a relatively simple numerical implementation, for the solution of the 'inverse' problem of finding the correct reconstruction of unknown profiles of chemical species within the sample, starting from experimental information gathered from micro-Raman depth profiling.

Improved maximum entropy method for the analysis of fluorescence spectroscopy data: evaluating zero-time shift and assessing its effect on the determination of fluorescence lifetimes.

A new algorithm based on the Maximum Entropy Method (MEM) is proposed for recovering both the lifetime distribution and the zero-time shift from time-resolved fluorescence decay intensities. The developed algorithm allows the analysis of complex time decays through an iterative scheme based on entropy maximization and the Brent method to determine the minimum of the reduced chi-squared value as a function of the zero-time shift. The accuracy of this algorithm has been assessed through comparisons with simulated fluorescence decays both of multi-exponential and broad lifetime distributions for different values of the zero-time shift. The method is capable of recovering the zero-time shift with an accuracy greater than 0.2% over a time range of 2000 ps. The center and the width of the lifetime distributions are retrieved with relative discrepancies that are lower than 0.1% and 1% for the multi-exponential and continuous lifetime distributions, respectively. The MEM algorithm is experimentally validated by applying the method to fluorescence measurements of the time decays of the flavin adenine dinucleotide (FAD).

Closed-form solution of the steady-state photon diffusion equation in the presence of absorbing inclusions.

We have developed a theoretical model for photon migration through scattering media in the presence of an absorbing inhomogeneity. A closed-form solution for the average diffuse intensity has been obtained through an iterative approximation scheme of the steady-state diffusion equation. The model describes absorbing defects in a wide range of values. Comparisons with the results of Monte Carlo simulations show that the error of the model is lower than 3% for size inclusion lower than 4 mm and absorption contrast up to the threshold value of the "black defect." The proposed model provides a tractable mathematical basis for diffuse optical and photoacoustic tomographic reconstruction techniques.

Detection of parathion pesticide by quartz crystal microbalance functionalized with UV-activated antibodies.

Photonic immobilization technique (PIT) has been used to develop an immunosensor for the detection of parathion. An antibody solution has been activated by breaking the disulfide bridge in the triad Trp/Cys-Cys through absorption of ultrashort UV laser pulses. The free thiol groups so produced interact with gold lamina making the antibody oriented upside, that is, with its variable parts exposed to the environment, thereby greatly increasing the detection efficiency. PIT has been applied to anchor polyclonal antiparathion antibodies to the gold electrode of a Quartz Crystal Microbalance (QCM) giving rise to very high detection sensitivity once the parathion is made heavier by complexion with BSA (bovine serum albumin), this latter step only required by the mass based transducer used in this case. The comparison of the sensor response with irradiated antibodies against different analytes shows that the high degree of antibody specificity is not affected by PIT nor is it by the complexion of parathion with BSA. These results pave the way to important applications in biosensing, since the widespread occurrence of the Trp/Cys-Cys residues triads in proteins make our procedure very general and effective to detect light analytes.

Ultraviolet laser-induced cross-linking in peptides.

RATIONALE: The aim of this study was to demonstrate, and to characterize by high-resolution mass spectrometry that it is possible to preferentially induce covalent cross-links in peptides by using high-energy femtosecond ultraviolet (UV) laser pulses. The cross-link is readily formed only when aromatic amino acids are present in the peptide sequence. METHODS: Three peptides, xenopsin, angiotensin I, and interleukin, individually or in combination, were exposed to high-energy femtosecond UV laser pulses, either alone or in the presence of spin trapping molecules, the reaction products being characterized by high resolution mass spectrometry. RESULTS: High-resolution mass spectrometry and spin trapping strategies showed that cross-linking occurs readily, proceeds via a radical mechanism, and is the highly dominant reaction, proceeding without causing significant photo-damage in the investigated range of experimental parameters. CONCLUSIONS: High-energy femtosecond UV laser pulses can be used to induce covalent cross-links between aromatic amino acids in peptides, overcoming photo-oxidation processes, that predominate as the mean laser pulse intensity approaches illumination conditions achievable with conventional UV light sources.

Analysis of simulated fluorescence intensities decays by a new maximum entropy method algorithm.

A new algorithm for the Maximum Entropy Method (MEM) is proposed for recovering the lifetime distribution in time-resolved fluorescence decays. The procedure is based on seeking the distribution that maximizes the Skilling entropy function subjected to the chi-squared constraint χ(2) ~ 1 through iterative linear approximations, LU decomposition of the Hessian matrix of the lagrangian problem and the Golden Section Search for backtracking. The accuracy of this algorithm has been investigated through comparisons with simulated fluorescence decays both of narrow and broad lifetime distributions. The proposed approach is capable to analyse datasets of up to 4,096 points with a discretization ranging from 100 to 1,000 lifetimes. A good agreement with non linear fitting estimates has been observed when the method has been applied to multi-exponential decays. Remarkable results have been also obtained for the broad lifetime distributions where the position is recovered with high accuracy and the distribution width is estimated within 3%. These results indicate that the procedure proposed generates MEM lifetime distributions that can be used to quantify the real heterogeneity of lifetimes in a sample.

Time-resolved flavin adenine dinucleotide fluorescence study of the interaction between immobilized glucose oxidase and glucose.

Time-resolved fluorescence experiments have shown that flavin adenine dinucleotide (FAD) fluorescence emission of sol-gel immobilized glucose oxidase (GOD) exhibits a three-exponential decaying behaviour characterized by long- (about 2.0-3.0 ns), intermediate- (about 300 ps) and short- (less than 10 ps) lifetime, each one being characteristic of a peculiar conformational state of the FAD structure. In the present work time-resolved fluorescence is used to monitor FAD signals in the time interval immediately following the addition of glucose at various concentrations in order to detect the conformational changes occurring during the interaction between sol-gel immobilized GOD and glucose. The analysis of time-dependent fluorescence emission signal has shown that the FAD conformational state changes during the process from a configuration with a prevalence of the state characterized by the long lifetime to a configuration with increased contribution from the process with the intermediate lifetime. The time needed to complete this configuration change decreases with the concentration of added glucose. The results here reported indicate that time-resoled fluorescence can be extremely useful for a better understanding of solid phase biocatalysis that is particularly important in light of their clinical and biotechnological applications.

Development and Validation of the Interpersonal Motivational Systems Questionnaire (IMS-Q).

Framed within the evolutionary framework, the Interpersonal Motivational System (IMS) theory suggests that eight distinct motivational impulses drive interpersonal human relationships, namely caregiving, social affiliation, attachment, rank-dominance, rank-submission, social play, cooperation, and sexuality. This theory has been widely applied in clinical practice, where psychopathology is viewed as the result of non-flexible or excessive activation of one system over another. Despite its clinical relevance, empirical studies aimed at measuring IMSs are scarce. This paper contributed to filling this gap by proposing a questionnaire to measure individuals' activation of the eight IMSs. Two studies involving large samples of adults were conducted. The first study (N = 455; 76.5% females) concerned the development of the questionnaire and examination of its content validity through explorative factor analysis. In the second study (N = 635; 54.8% females), confirmatory factor analyses were performed to further refine and confirm the instrument's factor structure. The final version consisted of 50 items. Empirical validity was established by investigating the correlations between the eight IMSs and other related measures (i.e., personality traits, human basic values, and attachment dimensions). The findings suggest that the IMS framework can be used to understand individual differences in motivation and behavior in different social contexts.

Spatially resolved CO2 carbon stable isotope analyses at the microscale using Raman spectroscopy.

Measuring the carbon stable isotope ratio (13C/12C, expressed as δ13CCO2) in geogenic CO2 fluids is a crucial geochemical tool for studying Earth's degassing. Carbon stable isotope analysis is traditionally performed by bulk mass spectrometry. Although Raman spectroscopy distinguishes 12CO2 and 13CO2 isotopologue bands in spectra, using this technique to determine CO2 isotopic signature has been challenging. Here, we report on in-situ non-destructive analyses of the C stable isotopic composition of CO2, applying a novel high-resolution Raman configuration on 42 high-density CO2 fluid inclusions in mantle rocks from the Lake Tana region (Ethiopia) and El Hierro (Canary Islands). We collected two sets of three spectra with different acquisition times at high spectral resolution in each fluid inclusion. Among the 84 sets of spectra, 58 were characterised by integrated 13CO2/12CO2 band area ratios with reproducibility better than 4‰. Our results demonstrate the determination of δ13CCO2 by Raman spectroscopy in individual fluid inclusions with an error better than 2.5 ‰, which satisfactorily matches bulk mass spectrometry analyses in the same rock samples, supporting the accuracy of the measurements. We thus show that Raman Spectroscopy can provide a fundamental methodology for non-destructive, site-specific, and spatially resolved carbon isotope labelling at the microscale.

Glucose sensing by time-resolved fluorescence of sol-gel immobilized glucose oxidase.

A monolithic silica gel matrix with entrapped glucose oxidase (GOD) was constructed as a bioactive element in an optical biosensor for glucose determination. Intrinsic fluorescence of free and immobilised GOD was investigated in the visible range in presence of different glucose concentrations by time-resolved spectroscopy with time-correlated single-photon counting detector. A three-exponential model was used for analysing the fluorescence transients. Fractional intensities and mean lifetime were shown to be sensitive to the enzymatic reaction and were used for obtaining calibration curve for glucose concentration determination. The sensing system proposed achieved high resolution (up to 0.17 mM) glucose determination with a detection range from 0.4 mM to 5 mM.

Depth dependence of estimated optical properties of a scattering inclusion by time-resolved contrast functions.

In this study we have theoretically and experimentally investigated the behavior of first order approximation contrast function when purely scattering inhomogeneities located at different depths inside a turbid thick slab are considered. Results of model predictions have been compared with Finite element method simulations and tested on phantoms. To this aim, we have developed for the first time to our knowledge a fitting algorithm for estimating both the scattering perturbation parameter and the shift of the inhomogeneity from the middle plane, allowing one to reduce the uncertainties due to depth. This is important for optical mammography because effects of the depth can cause uncertainties in the derived tumor optical properties that are above 20% and the scattering properties of tumors differ from those of the sourrounding healthy tissue by a comparable extent.

Modeling Retrograde Vitrification in the Polystyrene-Toluene System.

Atactic polystyrene, as reported in a recent contribution by our group, displays a marked change in glass transition when exposed to toluene vapor due to plasticization associated with vapor sorption within the polymer. The dependence of the glass transition temperature of the polymer-penetrant mixture on the pressure of toluene vapor is characterized by the so-called "retrograde vitrification" phenomenon, in that, at a constant pressure, a rubber to glass transition occurs by increasing the temperature. In this contribution, we have used a theoretical approach, based on the nonrandom lattice fluid thermodynamic model for the polymer-toluene mixture, to predict the state of this system, i.e., rubbery or glassy, as a function of fluid pressure and system temperature. The experimentally detectable glass transition is assumed to be a kinetically affected evidence of an underlying II order thermodynamic transition of the polymer mixture. On the basis of this hypothesis, the Gibbs-Di Marzio criterion, stating that equilibrium configurational entropy is zeroed at the glass transition, has been applied to locate the transition. The working set of equations consists of the expression of configurational entropy obtained from the adopted lattice fluid model equated to zero, coupled with the equation expressing the phase equilibrium between the polymer phase and the pure toluene vapor phase in contact and with the equations of state for the two phases. Theoretical predictions are in good qualitative and quantitative agreement with the experimental results previously obtained gravimetrically performing "dynamic" sorption experiments, which represent a neat example of the occurrence of so-called "type IV" glass transition temperature vs pressure behavior. The peculiar retrograde vitrification phenomenon and the glass transition temperature vs pressure envelope determined experimentally are well described by the proposed theoretical approach.

Glucose Determination by Means of Steady-state and Time-course UV Fluorescence in Free or Immobilized Glucose Oxidase.

Changes in steady-state UV fluorescence emission from free or immobilizedglucose oxidase have been investigated as a function of glucose concentration.Immobilized GOD has been obtained by entrapment into a gelatine membrane. Changes insteady-state UV fluorescence have been quantitatively characterized by means ofoptokinetic parameters and their values have been compared with those previouslyobtained for FAD fluorescence in the visible range. The results confirmed that greatercalibration ranges are obtained from UV signals both for free and immobilized GOD inrespect to those obtained under visible fluorescence excitation. An alternative method tothe use UV fluorescence for glucose determination has been investigated by using timecourse measurements for monitoring the differential fluorescence of the redox forms of theFAD in GOD. Also in this case quantitative analysis have been carried out and acomparison with different experimental configurations has been performed. Time coarsemeasurements could be particularly useful for glucose monitoring in complex biologicalfluids in which the intrinsic UV fluorescence of GOD could be not specific by consideringthe presence of numerous proteins.

Light assisted antibody immobilization for bio-sensing.

Ultrashort UV pulses at 258 nm with repetition rate of 10 kHz have been used to irradiate buffer solution of antibody. The tryptophan residues strongly absorb this radiation thus becoming capable to disrupt the disulfide bridges located next to them. Due to their high reactivity the opened bridges can anchor a gold plate more efficiently than other sites of the macromolecule giving rise to preferential orientations of the variable part of the antibody. UV irradiation has been applied to anchor antiIgG antibody to the electrode of a Quartz Crystal Microbalance (QCM) that lends itself as a sensor, the antibody acting as the bio-receptor. An increase of the QCM sensitivity and of the linear range has been measured when the antibody is irradiated with UV laser pulses. The photo-induced reactions leading to disulfide bridge breakage have been analyzed by means of a chemical assay that confirms our explanation. The control of disulfide bridges by UV light paves the way to important applications for sensing purpose since cysteine in combination with tryptophan can act as a hook to link refractory bio-receptors to surfaces.

Single isolated attosecond pulse from multicycle lasers.

We propose a new experimental scheme to produce clean isolated pulses lasting a few hundreds of attoseconds. It is based on high harmonics generation and uses the polarization gating technique combined with the ionization dynamics and the spatial filtering provided by the three-dimensional field propagation. The proposed method is easy to implement, robust against laser parameter fluctuations, and shows to be effective up to a 25 fs pulse duration.

Perturbation approach to the time-resolved transmittance for a spatially varying scattering inclusion in a diffusive slab.

In the framework of the perturbation approach to the diffusion equation, an analytical expression is derived to describe the effects on the time-resolved transmittance due to the presence of a spatially varying scattering inclusion hidden inside a diffusive slab. This formula assumes that the reduced scattering coefficient of the inclusion is spatially Gaussian distributed and complements that obtained for the absorptive case. The accuracy and the application range of the perturbed transmittance are investigated through comparisons with the numerical solutions of the time-dependent diffusion equation given by using the finite-element method. The proposed perturbation model is validated through a fitting procedure that determines the relative error in retrieving the scattering perturbation parameter of the inclusion located at the midplane of the slab.