Ines Delfino

Optical investigation of the electron transfer protein azurin–gold nanoparticle system

The hybrid system obtained by conjugating the protein azurin, which is a very stable and well-described protein showing a unique interplay among its electron transfer and optical properties, with 20-nm sized gold nanoparticles has been investigated. Binding of azurin molecules to gold nanoparticle surface results in the red shift of the nanoparticle resonance plasmon band and in the quenching of the azurin single tryptophan fluorescence signal. These findings together with the estimate of the hydrodynamic radius of the composite, obtained by means of Dynamic Light Scattering, are consistent with the formation of a monolayer of protein molecules, with preserved natural folding, on nanoparticle surface. The fluorescence quenching of azurin bound molecules is explained by an energy transfer from protein to metal surface and it is discussed in terms of the involvement of the Az electron transfer route in the interaction of the protein with the nanoparticle.

EXPERIMENTAL TESTS OF DIFFERENT SOLUTIONS TO THE DIFFUSION EQUATION FOR OPTICAL CHARACTERIZATION OF SCATTERING MEDIA BY TIME-RESOLVED TRANSMITTANCE

A detailed investigation of the use of time-resolved trasmittance for the optical characterization of scattering media by use of different analytical solutions to the diffusion equation has been performed. A femtosecond Ti:sapphire laser working at 800 nm and a streak camera with a time resolution of a few picoseconds were employed. Different latex and Intralipid solutions as well as biological samples were investigated. Reduced scattering coefficients were evaluated, and good agreement with the Mie predictions was found. An estimation of the order of magnitude of the absorption coefficient was obtained for the low-absorbance samples examined. These studies confirm experimentally that time-resolved trasmittance can be employed usefully for evaluating s values of thick scattering samples when a proper theoretical description that takes into account realistic boundary conditions is used.

Investigation on Clarified Fruit Juice Composition by Using Visible Light Micro-Raman Spectroscopy

Liquid samples of clarified apple and apricot juices at different production stages were investigated using visible light micro-Raman spectroscopy in order to assess its potential in monitoring fruit juice production. As is well-known, pectin plays a strategic role in the production of clarified juice and the possibility of using Raman for its detection during production was therefore evaluated. The data analysis has enabled the clear identification of pectin. In particular, Raman spectra of apple juice samples from washed and crushed fruits revealed a peak at 845 cm-1 (typical of pectin) which disappears in the Raman spectra of depectinised samples. The fructose content was also revealed by the presence of four peaks at 823 cm-1, 872 cm-1, 918 cm-1 and 975 cm-1. In the case of apricot juice, several Raman fingerprints of β-carotene at 1008, 1159 and 1520 cm-1 were also highlighted. Present results resulted interesting for the exclusive use of optical methods for the quantitative determination of the above-mentioned substances in place of the biochemical assays generally used for this purpose, which are time consuming and require different chemical reagents for each of them.

Enzyme distribution and secondary structure of sol-gel immobilized glucose oxidase by micro-attenuated total reflection FT-IR spectroscopy.

Glucose oxidase (GOD) immobilized into sol-gel matrices was studied by using Micro-Attenuated Total Reflection Fourier Transform Infrared (micro-ATR FT-IR) spectroscopy in order to characterize enzyme distribution and secondary structure in systems with valuable potentialities in amperometric and optical biosensing. Spectra were acquired in the 4000-600 cm(-1) frequency region and the analysis of specific fingerprints in the FT-IR spectra evidenced that the enzyme was actually immobilized in the matrix. The enzyme spatial distribution was obtained by examining the amide I and amide II band region of spectra from defined sample positions. The deconvolution of the amide I band in terms of lorentzian functions provided information on the secondary structure of the immobilized GOD. By this approach a macroscopic preservation of GOD activity upon immobilization was evidenced along with the existence of some matrix sites with locally inactivated GOD. To our knowledge this is the first example of point-by-point characterization of conformational changes of immobilized enzyme by means of micro-ATR infrared spectroscopy, thus confirming that this technique can be usefully employed for a non- or minimally-invasive detailed micro-characterization of catalytic supports in order to improve their functionality.

Micro-Raman Spectroscopy and Univariate Analysis for Monitoring Disease Follow-Up

Micro-Raman spectroscopy is a very promising tool for medical applications, thanks to its sensitivity to subtle changes in the chemical and structural characteristics of biological specimens. To fully exploit these promises, building a method of data analysis properly suited for the case under study is crucial. Here, a linear or univariate approach using a R2 determination coefficient is proposed for discriminating Raman spectra even with small differences. The validity of the proposed approach has been tested using Raman spectra of high purity glucose solutions collected in the 600 to 1,600 cm−1 region and also from solutions with two known solutes at different concentrations. After this validation step, the proposed analysis has been applied to Raman spectra from oral human tissues affected by Pemphigus Vulgaris (PV), a rare life-threatening autoimmune disease, for monitoring disease follow-up. Raman spectra have been obtained in the wavenumber regions from 1,050 to 1,700 cm−1 and 2,700 to 3,200 cm−1 from tissues of patients at different stages of pathology (active PV, under therapy and PV in remission stage) as confirmed by histopathological and immunofluorescence analysis. Differences in the spectra depending on tissue illness stage have been detected at 1,150–1,250 cm−1 (amide III) and 1,420–1,450 cm−1 (CH3 deformation) regions and around 1,650 cm−1 (amide I) and 2,930 cm−1 (CH3 symmetric stretch). The analysis of tissue Raman spectra by the proposed univariate method has allowed us to effectively differentiate tissues at different stages of pathology.

Yeast cytochrome c integrated with electronic elements: a nanoscopic and spectroscopic study down to single-molecule level

Various aspects of redox protein integration with nano-electronic elements are addressed by a multi-technique investigation of different yeast cytochrome c (YCC)-based hybrid systems. Three different immobilization strategies on gold via organic linkers are explored, involving either covalent bonding or electrostatic interaction. Specifically, Au surfaces are chemically modified by self-assembled monolayers (SAMs) exposing thiol-reactive groups, or by acid-oxidized single-wall carbon nanotubes (SWNTs). Atomic force microscopy and scanning tunnelling microscopy are employed to characterize the morphology and the electronic properties of single YCC molecules adsorbed on the modified gold surfaces. In each hybrid system, the protein molecules are stably assembled, in a native configuration. A standing-up arrangement of YCC on SAMs is suggested, together with an enhancement of the molecular conduction, as compared to YCC directly assembled on gold. The electrostatic interaction with functionalized SWNTs allows several YCC adsorption geometries, with a preferential high-spin haem configuration, as outlined by Raman spectroscopy. Moreover, the conduction properties of YCC, explored in different YCC nanojunctions by conductive atomic force microscopy, indicate the effectiveness of electrical conduction through the molecule and its dependence on the electrode material. The joint employment of several techniques confirms the key role of a well-designed immobilization strategy, for optimizing biorecognition capabilities and electrical coupling with conductive substrates at the single-molecule level, as a starting point for advanced applications in nano-biotechnology.

Depth dependence of the analytical expression for the width of the point spread function (spatial resolution) in time-resolved transillumination

Simple analytical expressions for the point spread function (PSF) at different depths can save computation time and improve the performance of inverse algorithms for optical imaging. In particular, application of such formulas simplifies quantification of the optical characteristics of tissue abnormalities inside highly scattering media. Earlier it was shown within the random walk theory framework that the PSF for time-resolved transillumination imaging through a highly scattering slab is well represented by a Gaussian. We have experimentally validated a simple formula of the random walk model for the effective width of this Gaussian, as a function of time delay, at different depths of the target. Presented analysis of published experimental data, concerning effective width of the PSF, for a slab of considerably smaller thickness also demonstrates good agreement between the data and predictions of our model. This PSF width determines spatial resolution of the time-resolved imaging and is widely discussed in the literature.

Visible micro-Raman spectroscopy of single human mammary epithelial cells exposed to x-ray radiation.

Abstract. A micro-Raman spectroscopy investigation has been performed in vitro on single human mammary epithelial cells after irradiation by graded x-ray doses. The analysis by principal component analysis (PCA) and interval-PCA (i-PCA) methods has allowed us to point out the small differences in the Raman spectra induced by irradiation. This experimental approach has enabled us to delineate radiation-induced changes in protein, nucleic acid, lipid, and carbohydrate content. In particular, the dose dependence of PCA and i-PCA components has been analyzed. Our results have confirmed that micro-Raman spectroscopy coupled to properly chosen data analysis methods is a very sensitive technique to detect early molecular changes at the single-cell level following exposure to ionizing radiation. This would help in developing innovative approaches to monitor radiation cancer radiotherapy outcome so as to reduce the overall radiation dose and minimize damage to the surrounding healthy cells, both aspects being of great importance in the field of radiation therapy.

Light scattering methods for tracking gold nanoparticles aggregation induced by biotin–neutravidin interaction

Dynamic and Static Light Scattering (DLS and SLS), Resonance Light Scattering (RLS) and angular-ratiometric methods have been used for investigating the aggregation of biotinylated bovine serum albumin gold nanoparticles induced by the interaction of biotin with its partner neutravidin. All the approaches have shown to be sensitive to the presence of neutravidin in solution at nM-concentrations. Changes in scattered light intensity dependence on the scattering angle (studied by SLS) and on the wavelength of the incident light (by RLS) have been observed. Predictions from Mie theory have enabled to connect these changes to the increase in the hydrodynamic size of the aggregates, quantified by DLS. The results here reported confirm the potentials of light scattering approach for realizing methods for analyte quantification and offer a good starting point for evaluating the limits and advantages of each method, which would further widen the use of light scattering approach in biosensing.

Determination of glucose content by means of visible micro-Raman spectroscopy and interval partial least square multivariate analysis

Micro-Raman spectroscopy and interval Partial Least Square (iPLS) multivariate analysis have been used for determining glucose concentration in various commercial sport drinks. By employing a visible excitation light (633 nm), micro-Raman spectra in the 600–1600 cm−1 wavenumber shift region have been recorded, showing well defined and separated vibrational fingerprints of the various contained sugars (glucose, fructose and sucrose). Glucose content was quantified by using the iPLS analysis based on a model built by employing aqueous glucose solutions as reference samples. The estimated glucose concentrations are in good agreement with the values obtained by using a biochemical assay. These results represent a significant step towards the development of a fast, simple, cost-effective Raman-based method for glucose quantification in products of food and beverage industry, alternative to expensive, time-, sample- and chemicals-consuming biochemical assays currently used in production and quality control processes.