Single-Cell Photothermal Analysis Induced by MoS2 Nanoparticles by Raman Spectroscopy

: Two-dimensional nanomaterials, such as MoS2 nanosheets, have been attracting increasing attention in cancer diagnosis and treatment, thanks to their peculiar physical and chemical properties. Although the mechanisms which regulate the interaction between these nanomaterials and cells are not yet completely understood, many studies have proved their efficient use in the photothermal treatment of cancer, and the response to MoS2 nanosheets at the single-cell level is less investigated. Clearly, this information can help in shedding light on the subtle cellular mechanisms ruling the interaction of this 2D material with cells and, eventually, to its cytotoxicity. In this study, we use confocal micro-Raman spectroscopy to reconstruct the thermal map of single cells targeted with MoS2 under continuous laser irradiation. The experiment is performed by analyzing the water O-H stretching band around 3,400 cm-1 whose tetrahedral structure is sensitive to the molecular environment and temperature. Compared to fluorescence-based approaches, this Raman-based strategy for temperature measurement does not suffer fluorophore instability, which can be significant under continuous laser irradiation. We demonstrate that irradiation of human breast cancer MCF7 cells targeted with MoS2 nanosheets causes a relevant photothermal effect, which is particularly high in the presence of MoS2 nanosheet aggregates. Laser-induced heating is strongly localized near such particles which, in turn, tend to accumulate near the cytoplasmic membrane. Globally, our experimental outcomes are expected to be important for tuning the nanosheet fabrication process

Spectroscopic properties of two 5′-(4-dimethylamino)azobenzene conjugated G-quadruplex forming oligonucleotides

The synthesis of two 5'-end (4-dimethylamino)azobenzene conjugated G-quadruplex forming aptamers, the thrombin binding aptamer (TBA) and the HIV-1 integrase aptamer (T30695), was performed. Their structural behavior was investigated by means of UV, CD, fluorescence spectroscopy, and gel electrophoresis techniques in K+-containing buffers and water-ethanol blends. Particularly, we observed that the presence of the 5'-(4-dimethylamino)azobenzene moiety leads TBA to form multimers instead of the typical monomolecular chair-like G-quadruplex and almost hampers T30695 G-quadruplex monomers to dimerize. Fluorescence studies evidenced that both the conjugated G-quadruplexes possess unique fluorescence features when excited at wavelengths corresponding to the UV absorption of the conjugated moiety. Furthermore, a preliminary investigation of the trans-cis conversion of the dye incorporated at the 5'-end of TBA and T30695 showed that, unlike the free dye, in K+-containing water-ethanol-triethylamine blend the trans-to-cis conversion was almost undetectable by means of a standard UV spectrophotometer

A systematic study of the valence electronic structure of cyclo(Gly–Phe), cyclo(Trp–Tyr) and cyclo(Trp–Trp) dipeptides in the gas phase

The electronic energy levels of cyclo(glycine–phenylalanine), cyclo(tryptophan–tyrosine) and cyclo(tryptophan–tryptophan) dipeptides are investigated with a joint experimental and theoretical approach. Experimentally, valence photoelectron spectra in the gas phase are measured using VUV radiation. Theoretically, we first obtain low-energy conformers through an automated conformer–rotamer ensemble sampling scheme based on tight-binding simulations. Then, different first principles computational schemes are considered to simulate the spectra: Hartree–Fock (HF), density functional theory (DFT) within the B3LYP approximation, the quasi-particle GW correction, and the quantumchemistry CCSD method. Theory allows assignment of the main features of the spectra. A discussion on the role of electronic correlation is provided, by comparing computationally cheaper DFT scheme (and GW) results with the accurate CCSD method

Biological interactions of biocompatible and water-dispersed MoS2 nanosheets with bacteria and human cells

Two dimensional materials beyond graphene such as MoS2 and WS2 are novel and interesting class of materials whose unique physico-chemical properties can be exploited in applications ranging from leading edge nanoelectronics to the frontiers between biomedicine and biotechnology. To unravel the potential of TMD crystals in biomedicine, control over their production through green and scalable routes in biocompatible solvents is critically important. Furthermore, considering multiple applications of eco-friendly 2D dispersions and their potential impact onto live matter, their toxicity and antimicrobial activity still remain an open issue. Herein, we focus on the current demands of 2D TMDs and produce high-quality, few-layered and defect-free MoS2 nanosheets, exfoliated and dispersed in pure water, stabilized up to three weeks. Hence, we studied the impact of this material on human cells by investigating its interactions with three cell lines: two tumoral, MCF7 (breast cancer) and U937 (leukemia), and one normal, HaCaT (epithelium). We observed novel and intriguing results, exhibiting evident cytotoxic effect induced in the tumor cell lines, absent in the normal cells in the tested conditions. The antibacterial action of MoS2 nanosheets is then investigated against a very dangerous gram negative bacterium, such as two types of Salmonellas: ATCC 14028 and wild-type Salmonella typhimurium. Additionally, concentration and layer-dependent modulation of cytotoxic effect is found both on human cells and Salmonellas

Revealing Biomolecules Dynamics by UV Ultrafast Spectroscopy

Establishing a stable covalent bond between proteins and nucleic acids, in molecular biology usually referred to as crosslinking, is a fundamental tool for the identification of the partners in the DNA-protein interaction, the latter being a vital biological process. Crosslinking with fs-UV lasers has been presented in the literature as a revolutionary technique to increase the otherwise low process yield of conventional methods based on chemical catalysts, conventional UV sources, or longer UV pulses. It is known that crosslinking induced in cells by ultrashort laser pulses has a twofold advantage over conventional methods: (i) it binds only species that are in proximity ("zero length" covalent bond) of the absorbed photons rather than favoring unspecific bonds amongst many possible species in the cell and (ii) it only takes place until the radiation is incident on the sample, thus paving the way for time-resolved studies of transient interactions. UV-based cross-link relies upon the large absorption cross section of DNA base in the UV region, although, good for our health, the probability for these photo-induced changes is not as big as the excitation rate since many of the photo-excited species will relax into the ground state in an ultrafast time scale, preventing any change or damage. Using femtosecond lasers is a real step ahead to make the biomolecules photo-react in a reasonable amount, but they also offer the unique possibility for time-resolved measurements thereby allowing the investigation of the basic mechanisms following the photo-activation. While irradiation with a relatively high intensity UV laser greatly increases the efficiency of protein-nucleic acid cross-linking, it is difficult, however, to investigate cross-linking in the presence of very complex molecules, namely DNA and protein, themselves. Therefore, in a reductionist approach, to mimic the bond formation between the DNA base and the nearby proteins the photocyclization in 5-Benzyluracil (5BU) has been proposed as a model system of crosslink reactions, in view of the simultaneous presence of the Uracil and Benzene playing the role of the DNA base and the aromatic residue of a protein, respectively. In order to design an experiment aimed at the measurement of the dynamics of photocyclization, we studied the steady-state absorption and emission (fluorescence) properties of 5BU and 5,6-benzyluracil (5,6BU), the latter being produced by exposing 5BU to UV ultrashort laser pulses. We found that after some time we can assume that all 5BU is completely transformed into 5,6BU and modifications in the absorption and fluorescence spectrum, fluorescence anisotropy, fluorescence quantum yields, and excited state lifetimes are observed when 5BU is photocyclized thus becoming 5,6BU. The high value of anisotropy in 5BU indicates that a large fraction of its fluorescence signal has a very short lifetime, in the range of few picoseconds; whereas in the case of 5,6BU, the main part of fluorescence has a much longer lifetime in the range of; few nanoseconds. The role of solvent is also studied in the photocyclization process and, in particular, we show that using water as the solvent, photocyclization rate is larger than that observed when methanol is used. Such a finding can be explained in terms of different configuration 5BU molecules assume in the solution, thereby providing hints on the paths leading to 5,6BU formation. Time-resolved 5BU fluorescence has been measured in nanosecond and femtosecond regimes by "Time Correlated Single Photon Counting" and "Fluorescence up-conversion" techniques, respectively, finding a very good agreement with a theoretical model (Molecular dynamics modeling). The results described so far allows one to design a possible experiment in which in a pump-probe scheme the photocyclization process can be followed in time. In such a setup an ultrashort UV laser pulse has to be split into two replica: One is used to trigger the interaction (excite the sample and start the photocyclization, for instance); the second pulse is then properly delayed to probe an optical observable like absorption or fluorescence, and eventually monitor the reaction. Of course, laser pulse properties, especially the temporal characteristics, are then important in determining the time resolution of such experiments. A common technique to measure the temporal duration of ultrashort pulses is based on nonlinear phenomena such as second-harmonic-generation; one of the most used experimental schemes is based on noncollinear autocorrelation. Unfortunately, such an approach is not feasible in the UV frequency range because of no crystal allows light propagation in the deep UV. Thus, we used an alternative nonlinear process in autocorrelation measurements: an autocorrelator is set up based on Two-Photon Absorption (TPA). The TPA signal has been measured and the information on the pulse duration has been retrieved form the FWHM of the fitted function. A deeper knowledge of the processes occurring in biomolecules and biological samples, as those reported in this thesis, might enable us to engineer them by preventing the undesirable ones or increasing the rate for the preferred routes

Temporal and spectral characterization of femtosecond deep-UV chirped pulses

In contrast to the case of pulses in the infrared (IR) and visible range, the temporal characterization of deep-UV femtosecond pulses in combination with their spectral features is still a challenge, essentially due to the lack of suitable nonlinear crystals for second harmonic autocorrelation. Here we report on the characterization of 260 nm, nearly 200 fs pulses, based on two photon absorption in fused silica. 260 nm pulses are obtained as the fourth harmonic component of a near-IR fundamental which is frequency up-converted into a double beta barium borate-based harmonic generator stage. By comparing the obtained pulse duration with its Fourier limit, estimated by measuring pulse spectra, a consistent pulse chirp is retrieved. This chirp is mostly attributed to the considerable group-velocity dispersion occurring in the last doubling stage which converts the green into UV radiation. Additionally, the spectral width of the probe pulse through the fused silica window turns out to be modulated as a function of the time delay between pump and probe in the two-photon absorption setup. The observed modulation is attributed to the interplay between spectrally selective absorption, due to the chirp of the pulses, and moderate self-phase modulation just occurring at the top of the temporal autocorrelation between pump and probe

Effect of different solvent condition on cis-trans isomerization of two benzodiazopyrrole derivatives

The trans-to-cis photo-isomerization of azobenzene (AzB) and its derivatives (AzDs) is a well known process (1), that, in the course of the last two decades has been studied extensively for its high potential in a wide range of technological applications, extending from the photo-control of biological events (2) to the production of engineered materials (3). In this frame, we synthesized and studied the chemical-physical properties of two benzodiazopyrrole derivatives, named 1RS and 1RR (Figure 1). The photo-chemical properties of both molecules were studied, by 1H NMR, UV, and fast UV spectroscopy, in dark and under irradiation by LED light at 435 nm, using different solvent conditions. In methanol, UV spectra registered on samples of 1RS or 1RR kept in dark show the highest band centered at 400 nm. After irradiation, the shape and the intensity of this band strongly changed, thus evidencing that trans-cis conversion should occur in the solution. 1H NMR spectra registered before and after irradiation also show significant differences, attributable to light induced trans-cis conversion. Fast UV experiments were used to measure the time required to convert trans 1RS and 1RR to the corresponding metastable cis isomers and the half lives of the latter species. Using aqueous solutions, the half lives of both metastable cis form strongly depend on the pH of the solution. In particular the half lives of both cis 1RR and 1RS increase from ~1s at pH=5.7 to > 90s at pH=8. Notably, increasing the LED light exposition of the samples, the UV profile of 1RR in buffered solution at pH=5.7, but not that of 1RS, undergoes to irreversible change in the intensity and the position of bands. We are now studying the origin of this phenomenon

Periodic Surface Structuring of Copper with Spherical and Cylindrical Lenses

The use of a cylindrical lens in femtosecond laser surface structuring is receiving attention to improve the processing efficiency. Here, we investigate the structures produced on a copper target, in air, by exploiting both spherical and cylindrical lenses for beam focusing, aiming at elucidating similarities and differences of the two approaches. The morphological features of the surface structures generated by ≈180 fs laser pulses at 1030 nm over areas of 8 × 8 mm2 were analyzed. For the spherical lens, micron-sized parallel channels are formed on the target surface, which is covered by subwavelength ripples and nanoparticles. Instead, the cylindrical lens leads to a surface decorated with ripples and nanoparticles with a negligible presence of micro-channels. Moreover, the morphological features achieved by focusing ≈180 fs laser pulses at 515 nm with the cylindrical lens and varying the scanning parameters were also studied. The experimental results evidence a direct effect of the hatch distance used in the scanning process on the target surface that contains dark and bright bands corresponding to regions where the rippled surface contains a richer decoration or a negligible redeposition of nanoparticles. Our findings can be of interest in large area surface structuring for the selection of the more appropriate focusing configuration according to the final application of the structured surface

Electrostatically driven scalable synthesis of MoS2–graphene hybrid films assisted by hydrophobins

Green synthesis of MoS2/biofunctionalized graphene hybrid films assisted by Vmh2 hydrophobin for applications in biosensing and photodetection