Vibrational spectroscopies for biochemical investigation of X-ray exposure effects on SH-SY5Y human neuroblastoma cells.

Neuroblastoma is the most recurring cancer in childhood and adolescence. The SH-SY5Y neuroblastoma cell line is generally adopted for elaborating new therapeutical approaches and/or elaborating strategies for the prevention of central nervous system disturbances. In fact, it represents a valid model system for investigating in vitro the effects on the brain of X-ray exposure using vibrational spectroscopies that can detect early radiation-induced molecular alterations of potential clinical usefulness. In recent years, we dedicated significant efforts in the use of Fourier-transform and Raman microspectroscopy techniques for characterizing such radiation-induced effects on SH-SY5Y cells by examining the contributions from different cell components (DNA, proteins, lipids, and carbohydrates) to the vibrational spectra. In this review, we aim at revising and comparing the main results of our studies to provide a wide outlook of the latest outcomes and a framework for future radiobiology research using vibrational spectroscopies. A short description of our experimental approaches and data analysis procedures is also reported.

Optical Sensors Technology and Applications.

Optical methods are non-invasive tools, and their use in various fields, including sensing applications, is continuously increasing, which is thanks to the continuous development of innovative low-cost sources and detectors [...].

Advanced Optical Sensing of Phenolic Compounds for Environmental Applications.

Phenolic compounds are particularly dangerous due to their ability to remain in the environment for a long period of time and their toxic effects. They enter in the environment in different ways, such as waste from paper manufacturing, agriculture (pesticides, insecticides, herbicides), pharmaceuticals, the petrochemical industry, and coal processing. Conventional methods for phenolic compounds detection present some disadvantages, such as cumbersome sample preparation, complex and time-consuming procedures, and need of expensive equipment. Therefore, there is a very large interest in developing sensors and new sensing schemes for fast and easy-to-use methods for detecting and monitoring the phenolic compound concentration in the environment, with special attention to water. Good analytical properties, reliability, and adaptability are required for the developed sensors. The present paper aims at revising the most generally used optical methods for designing and fabricating biosensors and sensors for phenolic compounds. Some selected examples of the most interesting applications of these techniques are also proposed.

Prebiotic Organic Chemistry of Formamide and the Origin of Life in Planetary Conditions: What We Know and What Is the Future.

The goal of prebiotic chemistry is the depiction of molecular evolution events preceding the emergence of life on Earth or elsewhere in the cosmos. Plausible experimental models require geochemical scenarios and robust chemistry. Today we know that the chemical and physical conditions for life to flourish on Earth were at work much earlier than thought, i.e., earlier than 4.4 billion years ago. In recent years, a geochemical model for the first five hundred million years of the history of our planet has been devised that would work as a cradle for life. Serpentinization processes in the Hadean eon affording self-assembled structures and vesicles provides the link between the catalytic properties of the inorganic environment and the impressive chemical potential of formamide to produce complete panels of organic molecules relevant in pre-genetic and pre-metabolic processes. Based on an interdisciplinary approach, we propose basic transformations connecting geochemistry to the chemistry of formamide, and we hint at the possible extension of this perspective to other worlds.

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.

Multivariate Analysis of Difference Raman Spectra of the Irradiated Nucleus and Cytoplasm Region of SH-SY5Y Human Neuroblastoma Cells.

Previous works showed that spatially resolved Raman spectra of cytoplasm and nucleus region of single cells exposed to X-rays evidence different features. The present work aims to introduce a new approach to profit from these differences to deeper investigate X-ray irradiation effects on single SH-SY5Y human neuroblastoma cells. For this aim, Raman micro-spectroscopy was performed in vitro on single cells after irradiation by graded X-ray doses (2, 4, 6, 8 Gy). Spectra from nucleus and cytoplasm regions were selectively acquired. The examination by interval Principal Component Analysis (i-PCA) of the difference spectra obtained by subtracting each cytoplasm-related spectrum from the corresponding one detected at the nucleus enabled us to reveal the subtle modifications of Raman features specific of different spatial cell regions. They were discussed in terms of effects induced by X-ray irradiation on DNA/RNA, lipids, and proteins. The proposed approach enabled us to evidence some features not outlined in previous investigations.

Graphene-Based Raman Spectroscopy for pH Sensing of X-rays Exposed and Unexposed Culture Media and Cells.

Graphene provides a unique way of sensing the local pH level of substances on the micrometric scale, with important implications for the monitoring of cellular metabolic activities where proton excretion could occur. Accordingly, an innovative biosensing approach for the quantification of the pH value of biological fluids, to be used also with small amounts of fluids, was realized and tested. It is based on the use of micro-Raman spectroscopy to detect the modifications of the graphene doping level induced by the contact of the graphene with the selected fluids. The approach was preliminarily tested on aqueous solutions of known pH values. It was then used to quantify the pH values of cell culture media directly exposed to different doses of X-ray radiation and to media exposed to X-ray-irradiated cells. The Raman response of cells placed on graphene layers was also examined.

Fructose and Pectin Detection in Fruit-Based Food Products by Surface-Enhanced Raman Spectroscopy.

Surface-Enhanced Raman Spectroscopy (SERS) enables the investigation of samples with weak specific Raman signals, such as opaque samples, including fruit juices and pulp. In this paper, biological apple juices and apple/pear pulp have been studied in order to evidence the presence of fructose and pectin, which are components of great relevance for quality assessment of these kinds of products. In order to perform SERS measurements a low-cost home-made substrate consisting of a glass slide decorated with 30-nm-sized gold nanoparticles has been designed and used. By employing a conventional micro-Raman spectroscopy set-up and a suitable data treatment based on "wavelet" denoising algorithms and background subtraction, spectra of pectin and fructose with clear Raman features have been obtained. The results have confirmed the potential of SERS in the food industry for product characterization, also considering the low-cost and the relative ease of the fabrication process of the employed SERS substrate.

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.

X-rays induced alterations in mechanical and biochemical properties of isolated SH-SY5Y nuclei.

BACKGROUND: The use of ionizing radiations in radiotherapy is an effective and very common cancer treatment after surgery. Although ionizing-radiation DNA damages are extensively investigated, little is known about their effects on the other nuclear components, since their variations when studied in whole cells can be difficult to decouple from those of the cytoplasmatic structures. The organization of nuclear components plays a functional role since they are directly involved in some of the nuclear response to chemical or physical stimuli. For this reason, studying the X-ray effects on nuclear components is a crucial step in radiobiology. MATERIALS AND METHODS: We have used Atomic Force Microscopy (AFM) and micro-FTIR to examine the biomechanical and biochemical properties of hydrated fixed nuclei isolated from neuroblastoma (SH-SY5Y) cells irradiated by 2, 4, 6 and 8 Gy X-ray doses. RESULTS: The experimental results have shown that, already at 2 Gy irradiation dose, the nuclei exhibit not only a DNA damage, but also relevant alterations of lipid saturation, protein secondary structure arrangement and a significant decrease in nuclear stiffness, which indicate a remarkable chromatin decondensation. CONCLUSIONS AND GENERAL SIGNIFICANCE: The present work demonstrates that a multi-technique approach, able to disclose multiple features, can be helpful to achieve a comprehensive picture of the X-ray irradiation effects of the nuclear components and distinguish them from those occurring at the level of cytoplasm.

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.

Green and Scalable Preparation of Colloidal Suspension of Lignin Nanoparticles and Its Application in Eco-friendly Sunscreen Formulations.

Lignin nanoparticles (LNPs) are applied in several industrial applications. The nanoprecipitation of LNPs is fast and inexpensive but currently still limited to the use of hazardous organic solvents, making it difficult to apply them on a large scale. Here, we report a scalable nanoprecipitation procedure for the preparation of colloidal lignin nanoparticles (cLNPs) by the use of the green solvents dimethylisosorbide and isopropylidene glycerol. Irrespective of the experimental conditions, cLNPs showed higher UV absorbing properties and radical scavenging activity than parent LNPs and raw lignin. cLNPs were successively used in the preparation of eco-friendly sunscreen formulations (SPF 15, 30, and 50+, as evaluated by the COLIPA assay), which showed high UV-shielding activity even in the absence of synthetic boosters (microplastics) and physical filters (TiO2 and ZnO). Biological assays on human HaCaT keratinocytes and human skin equivalents demonstrated the absence of cytotoxicity and genotoxicity, associated with an optimal protection of the skin from UV-A damage.

Application of Vibrational Spectroscopies in the Qualitative Analysis of Gingival Crevicular Fluid and Periodontal Ligament during Orthodontic Tooth Movement.

Optical vibrational techniques show a high potentiality in many biomedical fields for their characteristics of high sensitivity in revealing detailed information on composition, structure, and molecular interaction with reduced analysis time. In the last years, we have used these techniques for investigating gingival crevicular fluid (GCF) and periodontal ligament (PDL) during orthodontic tooth treatment. The analysis with Raman and infrared signals of GCF and PDL samples highlighted that different days of orthodontic force application causes modifications in the molecular secondary structure at specific wavenumbers related to the Amide I, Amide III, CH deformation, and CH3/CH2. In the present review, we report the most relevant results and a brief description of the experimental techniques and data analysis procedure in order to evidence that the vibrational spectroscopies could be a potential useful tool for an immediate monitoring of the individual patient's response to the orthodontic tooth movement, aiming to more personalized treatment reducing any side effects.

Tracking excited state decay mechanisms of pyrimidine nucleosides in real time.

DNA owes its remarkable photostability to its building blocks-the nucleosides-that efficiently dissipate the energy acquired upon ultraviolet light absorption. The mechanism occurring on a sub-picosecond time scale has been a matter of intense debate. Here we combine sub-30-fs transient absorption spectroscopy experiments with broad spectral coverage and state-of-the-art mixed quantum-classical dynamics with spectral signal simulations to resolve the early steps of the deactivation mechanisms of uridine (Urd) and 5-methyluridine (5mUrd) in aqueous solution. We track the wave packet motion from the Franck-Condon region to the conical intersections (CIs) with the ground state and observe spectral signatures of excited-state vibrational modes. 5mUrd exhibits an order of magnitude longer lifetime with respect to Urd due to the solvent reorganization needed to facilitate bulky methyl group motions leading to the CI. This activates potentially lesion-inducing dynamics such as ring opening. Involvement of the 1nπ* state is found to be negligible.

Surface-enhanced Raman spectroscopy of tears: toward a diagnostic tool for neurodegenerative disease identification.

SIGNIFICANCE: A noninvasive method based on surface-enhanced Raman spectroscopy (SERS) of tears was proposed as a support for diagnosing neurodegenerative pathologies, including different forms of dementia and Alzheimer's disease (AD). In this field, timely and reliable discrimination and diagnosis are critical aspects for choosing a valid medical therapy, and new methods are highly required. AIM: The aim is to evince spectral differences in SERS response of human tears from AD affected, mild cognitive impaired (MCI), and healthy control (Ctr) subjects. APPROACH: Human tears were characterized by SERS coupled with multivariate data analysis. Thirty-one informed subjects (Ctr, MCI, and AD) were considered. RESULTS: Average SERS spectra from Ctr, MCI, and AD subjects evidenced differences related to lactoferrin and lysozyme protein components. Quantitative changes were also observed by determining the intensity ratio between selected bands. We also constructed a classification model that discriminated among AD, MCI, and Ctr subjects. The model was built using the scores obtained by performing principal component analysis on specific spectral regions (i-PCA). CONCLUSIONS: The results are very encouraging with interesting perspectives for medical applications as support of clinical diagnosis and discrimination of AD from other forms of dementia.