Development of novel nano-structured materials for Enhanced Raman Spectroscopies: an insight in SERS and TERS applications

Owing to their ultrahigh selectivity and sensitivity, the plasmon-enhanced Raman spectroscopies (PERS) methods have emerged as diverse and exciting cutting-edge techniques for the investigation of biosystems at nanometric scales in air or water environments. By exploiting the plasmonic properties of noble metal nanoparticles, the PERS methods enable to remove the main obstacle of the Raman spectroscopy represented by the small Raman cross- section. Among them, the Surface Enhanced Raman Spectroscopy (SERS) is certainly the most important in terms of the number of applications in many fields of science (physics, chemistry, and biomedicine). One of the interesting features of the SERS is that the huge amplification of inelastic Raman photons can reach up to 12 orders of magnitude allowing even the detection at single-molecule level. In addition, the strong distance dependence of the plasmonic near-field effect (∼ 10-20 nm) makes effective the SERS only for molecules in proximity to metal-nanostructured surfaces and, thus, suitable not only for the bio-analysis of membranes but also for the surface characterization in science materials. Anyway, beyond the high sensitivity, the limitation of the SERS is represented by the diffracted-limited spatial resolution. A significant improvement is given by the modern tip-enhanced Raman spectroscopy (TERS) technique. By combining the high resolution of scanning-probe microscope (SPM) technology and the sensitivity of SERS, TERS is capable to correlate topographical and chemical information of a sample at nanoscale level. In fact, the Raman signal coming from the probed molecules is strongly enhanced via SERS effect when they are in proximity of the apex of a metalized or metallic SPM tips. Moreover, the scattering efficiency of TERS signal is greatly increased when the metal surface of the probe is nano-structured. The spatial resolution of TERS signals is mainly ruled by the tip-radius, which is typically of few tens of nanometers, therefore allowing to reach a lateral resolution in the range of 10-50 nm, far beyond the diffraction limit. Anyway, the development of reliable and effective plasmonic devices for SERS and TERS applications represents the major obstacle towards a wider diffusion of TERS/SERS as powerful analytical tools in material science and life science. In the case of TERS, the main technological challenge is based on the fabrication of metal nano-structures on the tip. Compared to SERS substrates that are produced on large-area surfaces, the sub-micron dimensions of the tip apex make the nano-structuring task more tricky. In this frame, the current thesis work aims to present a novel and versatile method for the preparation of appropriate AFM-TERS tips and SERS substrates. The innovative approach is based on the application of a radio-frequency discharge produced by an inductively coupled plasma (ICP) on commercial Ag-covered AFM probes. The plasma treatment produces an intriguing metallic porous nanotexture resembling a coral-like structure. The so-produced probes have been characterized by showing an amplification up to six orders of magnitude and a spatial resolution down to 10 nm, which render these devices particularly attractive for nanometer chemical characterization. In addition, this method has been successfully implemented for the fabrication of broad-band SERS-active platforms. This protocol has shown to be effective to produce substrates that can amplify the Raman signal up to seven orders of magnitude. Finally, another method for the fabrication of SERS substrates, based on the self-assembly of block copolymer (BCP) loaded with Ag-NPs, is proposed. The sensitivity of the so-prepared substrates has been tested by revealing the over-expression of target proteins in membranes of cancer cells

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

Epigenetic Silencing of Peroxisome Proliferator-Activated Receptor γ Is a Biomarker for Colorectal Cancer Progression and Adverse Patients' Outcome

The relationship between peroxisome proliferator-activated receptor γ (PPARG) expression and epigenetic changes occurring in colorectal-cancer pathogenesis is largely unknown. We investigated whether PPARG is epigenetically regulated in colorectal cancer (CRC) progression. PPARG expression was assessed in CRC tissues and paired normal mucosa by western blot and immunohistochemistry and related to patients' clinicopathological parameters and survival. PPARG promoter methylation was analyzed by methylation-specific-PCR and bisulphite sequencing. PPARG expression and promoter methylation were similarly examined also in CRC derived cell lines. Chromatin immunoprecipitation in basal conditions and after epigenetic treatment was performed along with knocking-down experiments of putative regulatory factors. Gene expression was monitored by immunoblotting and functional assays of cell proliferation and invasiveness. Methylation on a specific region of the promoter is strongly correlated with PPARG lack of expression in 30% of primary CRCs and with patients' poor prognosis. Remarkably, the same methylation pattern is found in PPARG-negative CRC cell lines. Epigenetic treatment with 5′-aza-2′-deoxycytidine can revert this condition and, in combination with trichostatin A, dramatically re-activates gene transcription and receptor activity. Transcriptional silencing is due to the recruitment of MeCP2, HDAC1 and EZH2 that impart repressive chromatin signatures determining an increased cell proliferative and invasive potential, features that can experimentally be reverted. Our findings provide a novel mechanistic insight into epigenetic silencing of PPARG in CRC that may be relevant as a prognostic marker of tumor progression

Raman Analysis of Tear Fluid Alteration Following Contact Lense Use

Tear fluid is a heterogeneous solution containing mainly proteins, lipids, mucins and electrolytes, which regulates the physiology of the human eye. The complex composition of tears can be altered in the presence of eye inflammations. The use of contact lenses is one of the most frequent causes of inflammatory responses of the eye, with the related discomfort often causing the wearer to give up using them. In this paper, we exploit the potentiality of Raman Spectroscopy to analyse the biochemical changes in tear fluid in a contact lens wearer. In particular, we analysed the tear fluid collected from a volunteer as a function of the wearing time for two types of monthly contact lenses (Hydrogel and Si-Hydrogel). Our experimental results show an alteration of the relative concentrations of proteins and lipids in both of the analysed cases. More importantly, our results highlight the diagnostic sensitivity of Raman analysis to select the proper contact lens type for each wearer and optimise the lens wearing conditions

Fabrication of Silver Coral-like AFM Probes for Tip-Enhanced Raman Spectroscopy by ICP-based approach

We describe a novel approach for the fabrication of plasmonic probes designed for Tip Enhanced Raman Spectroscopy (TERS) applications, based on the nano-structuration of Ag-coated commercial AFM probes via Inductively Coupled Plasma (ICP) discharge

Raman Analysis of Tear Fluid Alteration Following Contact Lense Use

Tear fluid is a heterogeneous solution containing mainly proteins, lipids, mucins and electrolytes, which regulates the physiology of the human eye. The complex composition of tears can be altered in the presence of eye inflammations. The use of contact lenses is one of the most frequent causes of inflammatory responses of the eye, with the related discomfort often causing the wearer to give up using them. In this paper, we exploit the potentiality of Raman Spectroscopy to analyse the biochemical changes in tear fluid in a contact lens wearer. In particular, we analysed the tear fluid collected from a volunteer as a function of the wearing time for two types of monthly contact lenses (Hydrogel and Si-Hydrogel). Our experimental results show an alteration of the relative concentrations of proteins and lipids in both of the analysed cases. More importantly, our results highlight the diagnostic sensitivity of Raman analysis to select the proper contact lens type for each wearer and optimise the lens wearing conditions

Revealing membrane alteration in cellsoverexpressing CA IX and EGFR by Surface-Enhanced Raman Scattering

Sensitive detection of altered proteins expression in plasma membranes is of fundamental importance, for both diagnostic and prognostic purposes. Surface-Enhanced Raman Scattering (SERS) has proven to be a quite sensitive approach to detect proteins, even in very diluted samples. However, proteins detection in complex environment, such as the cellular membrane, is still a challenge. Herein, we demonstrate a SERS-based platform to reveal the overexpression of target proteins in cell membranes. As a proof of concept, we implemented ectopic expression of carbonic anhydrase IX (CA IX) and epidermal growth factor receptor (EGFR) in the plasma membrane of the SKOV3 tumor cell line. Our outcomes demonstrate that SERS signals from cells put in contact with a hyperuniform SERS substrate allow highlighting subtle differences in the biochemical composition of cell membranes, normally hidden in spontaneous Raman confocal microscopy. This opens new opportunities for a label-free membrane analysis and bio-sensing in a broader sense