The ability to make an accurate diagnosis at the time of treatment is crucial for many diseases. However, current standard diagnostic procedures can only be performed in specialised healthcare facilities. To bring diagnostic methods from a specialised laboratory to the point of treatment, many alternative methods have been proposed. One of them is surfaceenhanced Raman scattering (SERS), which offers advantageous features such as high sensitivity in biotarget detection and higher accuracy. Here, we have developed an advanced SERS platform for the ultrasensitive, rapid and highly specific identification of tumour biomarkers in liquid biopsies. Our particular focus is on the detection of Thyroglobulin (Tg), the most important tumour biomarker for the diagnosis and prognosis of thyroid cancer. Specifically, SERS-active substrates fabricated by nanosphere lithography on chip or on tips of optical fiber (OF) were functionalized with Tg Capture antibodies. Gold nanoparticles were functionalized with Detection antibodies and conjugated with a Raman reporter. The sandwich assay platform was validated in the planar configuration and a detection limit of only 7 pg/ml was successfully achieved. The same approach has been successfully demonstrated on washout fluids from fine needle aspiration biopsies of cancer patients. Finally, the functionalization strategy was translated to the LOF-SERS platform and successfully used to detect Tg concentration. The proposed SERS-assisted immunoassay platform has proven to be highly versatile and can be used with both microfluidic chip POC devices and SERS-OF-based optrodes to perform sensitive, specific and rapid ex vivo assays for Tg detection in liquid intraoperative biopsies.
In this work, we propose a novel immunoassay platform for the detection of human Thyroglobulin (Tg) to be integrated with fine-needle aspiration biopsy for early identification of lymph node metastases in thyroid cancer patients. The sensing platform detects Tg (a well-known biomarker for the classification of metastatic lymph nodes related to thyroid cancer) by a sandwich immunoassay involving a self-assembled surface-enhanced Raman scattering (SERS) substrate assisted and empowered by functionalized gold nanoparticles enabling additional Raman signal amplification and improved molecular specificity. The sandwich assay platform was preliminary validated in a planar configuration and a detection limit as low as 7 pg/mL was successfully achieved. The sandwich assay was successfully demonstrated on washout fluids of fine needle aspiration biopsies from cancer patients and confirmed the high specificity of the proposed methodology when complex biological matrices are considered. Finally, optical fiber SERS optrodes were fabricated and successfully used to detect Tg concentration by applying the same bio-recognition strategy. This opens the possibility of transferring the Tg detection approach to the optical fiber tip to develop point-of-care platforms that can be directly integrated into fine needle aspiration biopsies.
In the last few years, gold nanoparticles (AuNPs) have been widely used in biomedical applications; unfortunately, in some cases, they induce toxic effects on biological systems. Among these, the activation of the innate immune system (inflammatory response) is considered a central issue for assessing health risks. Although the origin of nanotoxicity is not well known, the cause could be associated with the presence of contaminants on nanoparticles surface, such as bacterial endotoxin. The bacterial endotoxin, also known as Lipopolysaccharide (LPS), is the main component of gram-negative bacteria cell walls and is considered one of the major contaminants in the environment. Therefore, AuNPs can be easily contaminated during the synthesis process or their manipulation, and nanotoxicological assessment has to be performed before using AuNPs for biomedical purposes. Traditional assays for LPS detection are not always effective because AuNPs could interfere with other components and alter the final readout. Surface-enhanced Raman spectroscopy (SERS) is a powerful tool for the analysis of molecules on metallic NPs, allowing to detect low concentration LPS with excellent sensitivity and reproducibility. In this work, we demonstrate that SERS technique can detect low amount of LPS (fg) on AuNPs (50 nm); moreover, we studied the internalization of bare and LPS-coated AuNPs in human monocyte-derived macrophages, by Raman Imaging, and their inflammatory effect by in vitro evaluation of pro and anti-inflammatory cytokine production.
Here, we present a multimodal imaging approach based on Raman spectroscopy and PSDH imaging to detect circulating tumor cells. Particularly, the ability of tumor cells to internalize glucose faster than normal cells was investigated. Deuterated-glucose was used as Raman vibrational tag since its signal is present in the “silent” zone of Raman cell spectra. Raman information were correlated with the visualization of the cell birefringence by PSDH imaging. Each technique provides different information which combination gives a molecular and morphological signature of each cell, allowing to discriminate between normal and cancer cells with a sensitivity and specificity up to 80%.
Surface-enhanced Raman scattering (SERS) has established itself as powerful tool for molecular sensing in biology and medicine. The integration of SERS systems with optical fiber is a challenging but potentially very rewarding endeavour. However, efforts to transfer the technology from the laboratory to the clinic have been frustrated by the lack of robust stable and sensitive substrates on the fiber tip, as well as the complexity of interfacing between sample and the substrate itself. Here, we propose the Lab-on-Fiber SERS optrodes, realized on the optical fiber tip by nanosphere lithography. Three types of highly ordered and reproducible SERS-active substrates have been realized: close-packed array (CPA); CPA after sphere removal (SR) and sparse array (SA) of polystyrene nanospheres, covered by a gold thin layer. To optimize the SERS probes, we compared the SERS performances in terms of Enhancement Factor (EF) and reproducibility pertaining to different patterns with different nanosphere diameters and gold thicknesses using the biphenyl-4-thiol (BPT), as target molecule. Moreover, we analysed and compared the SERS spectra of two representative biological probes, bovine serum albumin (BSA, medium molecule) and red blood cells (RBCs), in order to correlate the SERS response to the morphology and hysteric hindrance of the biological target. The SERS analysis indicated that the CPA substrate amplifies the BPT Raman intensity twice as well as the SR and SA substrates, while BSA and RBCs, with the CPA substrate, provide signals comparable to those of SR and SA substrates. Finally, we have optimized a Raman system for SERS optrode operation with efficient lighting and collection via optical fiber.
We report on our activities related to the development of surface enhanced Raman scattering (SERS) probes realized onto the optical fiber tip (OFT) through nanosphere lithography. In the first stage of our research, we adapted the nanosphere lithography to operate on the optical fiber tip, by assessing the process and demonstrating either the potentiality or the repeatability of the proposed nanopatterning approach. Successively, we investigated the ability of the manufactured structures on the fiber tip to act as SERS probes by measuring the SERS spectra in presence of a Biphenyl Thiol (BPT) monolayer. Firstly, we focused the attention on the samples shaped as closed packed array of nanospheres covered by gold. The analysis allowed us to identify the most promising SERS platform, exhibiting an Enhancement Factor (EF) of 4×105 and a SERS measurements variability lower than 10%. We addressed also the limitations related to the use of the same optical fiber for both illumination and light collection by selecting a commercial optical fiber exhibiting a suitable trade-off in terms of high excitation/collection efficiency and low silica background. Current activities are devoted to the investigation of other nanopatterns on the optical fiber tip (namely, Sparse Array of metallodielectric spheres) and the analysis of the probes response against different molecules.
In this work, we employed the Raman microscopy to study the internalization kinetics and spatial distribution of small interfering RNA (siRNA)-diatomite nanoparticles (DNPs) complex in human lung epidermoid carcinoma cell line (H1355) up to 72 h. Raman images are compared with confocal fluorescence microscopy results. The Raman analysis provides that the siRNA-DNPs are internalized and co-localized in lipid vesicles within 18 h, after that equilibrium is achieved.
Metasurfaces are two-dimensional structures, arrays of scatterers with subwavelength separation or optically thin planar films, allowing light manipulation and enabling specific changes of optical properties, as for example beam-steering, anomalous refraction and optical-wavefront shaping. Due to the fabrication simplicity, the metasurfaces offer an alternative to 3-D metamaterials and providing a novel method for optical elements miniaturization. It has been demonstrated that a metasurface can support Bound States in Continuum (BIC), that are resonant states by zero width, due to the interaction between trapped electromagnetic. Experimentally, this involves very narrow coupled resonances, with a high Q-factor and an extremely large field intensity enhancement, up to 6 orders of magnitude larger than the intensity of the incident beam. Here, we demonstrate that the field enhancement in proximity of the surface can be applied to boost fluorescence emission of probe molecules dispersed on the surface of a photonic crystal membrane fabricated in silicon nitride. Our results provide new solutions for light manipulation at the nanoscale, especially for sensing and nonlinear optics applications.
Semen analysis is widely used as diagnostic tool for assessing male fertility, controlling and managing the animal reproduction. The most important parameters measured in a semen analysis are the morphology and biochemical alterations. For obtaining such information, non-invasive, label-free and non-destructive techniques have to be used. Digital Holography (DH) combined with Raman Spectroscopy (RS) could represent the perfect candidate for a rapid, non-destructive and high-sensitive morphological and biochemical sperm cell analysis. In this study, DH-RS combined approach is used for a complete analysis of single bovine spermatozoa. High-resolution images of bovine sperm have been obtained by DH microscopy from the reconstruction of a single acquired hologram, highlighting in some cases morphological alterations. Quantitative 3D reconstructions of sperm head, both normal and anomalous, have been studied and an unexpected structure of the post-acrosomal region of the head has been detected. Such anomalies have been also confirmed by Raman imaging analysis, suggesting the protein vibrations as associated Raman marker of the defect.
Currently, a combination of technologies is typically required to identify and classify leukemia cells. These methods often lack the specificity and sensitivity necessary for early and accurate diagnosis. Here, we demonstrate the use of Raman spectroscopy to identify normal B cells, collected from healthy patients, and three ALL cell lines (RS4;11, REH and MN60 at different differentiation level, respectively). Raman markers associated with DNA and protein vibrational modes have been identified that exhibit excellent discriminating power for leukemia cell identification. Principal Component Analysis was finally used to confirm the significance of these markers for identify leukemia cells and classifying the data. The obtained results indicate a sorting accuracy of 96% between the three leukemia cell lines.
We developed a SERS biosensor based on gold fishnets fabricated by using e-beam lithography. This device is used for glycerophosphoinositol (GroPIns) molecule sensing. GroPIns is an abundant component of cell cytosol and high GroPIns levels have been reported in several tumour cells. We demonstrate that our SERS sensor is able to accurately and quantitatively determine the concentration of GroPIns. These results indicate that SERS may provide a novel platform technology to identify GroPIns profiles in disease pathogenesis.
The current study relates to a Raman spectroscopy-based method for addressing the problem of sex assessment in
mammals. A direct method for sex predetermination in animals is based on the X- and Y-bearing sperm cells sorting
before insemination. Our Raman spectroscope allows distinguishing and characterizing the difference between X- and
Y-bearing sperm cells by detecting and analyzing their Raman spectra in a non-invasive and non-destructive way.
Raman spectroscopy is a label-free and non-invasive method that measures the inelastic scattered light from a sample
giving insight into the vibration eigenmodes of the excited molecules. For these reasons, Raman spectroscopy has been
used as a powerful tool to investigate different biological tissues and living cells. In this paper, we present a Raman
spectroscopy-based method for sensitive biochemical characterization of bovine sperm cells. Importantly, by analysing
separate Raman spectra from the nucleus, acrosomale vesicle and tail of single sperm cells, we are able to identify
characteristic Raman features associated with DNA, protein and lipid molecular vibrations for discriminating among
different locations inside the cell with sub-micrometric resolution (∼0.3 μm). We demonstrate that our Raman
spectroscopy facilitates spectral assignment and increases detection sensitivity, opening the way for novel bio-imaging
platforms.
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