SURFACE- AND TIP- ENHANCED RAMAN SPECTROSCOPY OF BIOMOLECULES

Surface- Enhanced Raman Scattering (SERS) and Tip-Enhanced Raman Scattering (TERS) exploit the enhancement of the local electromagnetic field induced by optical nanoantennas and nano-tips to largely amplify the Raman scattering of molecules located in the near-field of these structures, the so called hot-spots. With SERS or TERS, Raman scattering amplifications up to ten orders of magnitude can be obtained. SERS sensors based on nanoparticles of different materials and shapes have been developed for applications in the detection of pollutants, biomarkers, explosives, food pathogens, etc. Large area, high reproducibility, chemical stability, even flexibility, combined to high enhancement factors are key factors that are driving the research efforts in this field. TERS, in addition to SERS, permits to place and spatially control the position of the hot spot at the tip apex, allowing for imaging with nanoscale resolution or, under proper conditions, atomic scale resolution. Several TERS commercial setups are now available, coupling compact Raman spectrometers with with scanning probe microscopy (SPM) platforms, like atomic force microscopy (AFM), scanning tunneling microscopy (STM) or Shear-Force Microscopy (ShFM). These techniques can offer new original approaches in many fields, such as analytical chemistry, biology and biotechnology, forensic science and cultural heritage. One of the most appealing application field for SERS and TERS is biomolecular spectroscopy. Biomolecular sensors have become fundamental in biomedical research and the human health improvement. One of the crucial applications is in early stage diagnosis of diseases, through the detection, identification and quantification of some specific proteins, generally called biomarkers, that are present in very low quantities in body fluids (sub-nanomolar range). Immunoassays, such as enzyme-linked immunosorbent assay (ELISA), protein arrays, Western blots, immune-histochemistry and immune-fluorescence are well assessed probes The sensitivity is generally in the µM range, although new strategies have been proposed to achieve sub-femtomolar sensitivity. Moreover, these techniques are “indirect” methods, characterized by long operation times (several hours) and, especially at ultralow concentrations, can yield a large number of false positive detection events. Surface- and Tip- Enhanced Raman spectroscopies can tailor molecular sensitivity down to the atto-molar range, and to the single-molecule level in some particular configurations. Different concepts of SERS-based biosensors have been demonstrated so far. Raman dye-labeled sensors exploit SERS-active labels (NPs coated with high Raman cross-section dyes and functionalized with antibodies against the target molecule) to spot proteins, permitting their indirect detection (the signal of the dye is monitored) also in-vivo. Direct, label-free SERS sensors, in which the vibrational fingerprint of the target molecule is used for detection, are, however, desirable. This is due to operational rapidity, simplicity and richness of information embedded in the vibrational spectrum (e.g. on the functional state of a protein). Recently much effort has been addressed to SERS detection of biomolecules in liquid environment, i.e. their natural habitat. New tools for the manipulation of plasmonic nanoparticles in liquid environments like proteins buffered solutions, or in cells cultures are required for this task. The use of optical forces exerted by tightly focused laser beams on micro and nanostructures, is among the most promising emergent strategies for manipulation, control and SERS detection of molecular and biomolecular compounds in liquid. Optical forces can be used to attract or push micro and nano-objects from the focus of a lens (typically a microscope objective) in a contactless way. In particular, the use of the radiation pressure, has no restriction on excitation wavelength and permits to push nanoparticles along the beam direction, permitting to induce and control the formation of SERS-active aggregates in liquid. This latter point is yet largely unexplored among the scientific community. A crucial point for an efficient biosensor is the selective interaction of its active surface with biomolecules. In this framework the employment of bioreceptors increases the affinity of the sensor with the target molecule. Antibodies have been used in many SERS detection schemes for the functionalization of metal nanoparticles, but usually a Raman-active dye molecule is used as label for protein detection, due to the large dimensions of antibodies that prevent biomolecules to feel the plasmonic field enhancement. A very interesting alternative to antibodies is represented by DNA aptamers, whose nucleobases sequence can be properly designed for the specific binding with target biomolecules. Aptamers are more efficient and smaller with respect to antibodies, allowing for a direct characterization of the vibrational fingerprint of the biomolecules selectively linked to DNA strands. In addition chemical manipulation of aptamers is simpler, enabling the possibility to include a free thiol group at the end of DNA sequence for increasing the affinity with gold surfaces of SERS biosensors. Therefore the combination of the high sensibility and specificity within a label free approach, make SERS biosensors very appealing tools that can overcome the limitations of the conventional immunoassays in the way towards early diagnosis of cancer diseases. The work presented in this Thesis was focused, on one side, on the study of the basic electromagnetic processes in SERS, including polarization- issues, targeted at optimizing the excitation geometry of a nanosensor and, on the other side, on the development of new strategies for high sensitivity and specific detection of biomolecules in dry and liquid conditions by means of Surface- and Tip- enhanced Raman spectroscopy. In the initial part the attention has been focused on polarization issues related to the twofold electromagnetic enhancement mechanism in Surface enhanced Raman Scattering (SERS). We have discussed how the re-radiation effect can strongly alter the polarization state of the SERS radiation, influencing the SERS depolarization ratio. We have developed a model to relate the SERS depolarization to the molecular depolarization ratio, an intriguing physical quantity that provides information on the molecular orientation. Furthermore, we have studied, both theoretically and experimentally, the polarization properties of SERS from near-field coupled nanowires excited with circularly polarized light. From a practical point of view, this configuration turns out to be very attractive since the signal intensity becomes insensitive to the exact orientation of the sample, making sensors more robust to optical misalignments. Afterwards, driven by the necessity to develop new SERS sensors featuring large area, low cost, highly reproducibility and field enhancement, we have characterized SERS enhancement of novel nanostructures, namely Au nanocrescents evaporated over monolayers of polymeric nanospheres and asymmetric Au nanoclusters grown on flexible PDMS substrates. In both cases we have found a signal amplification up to four orders of magnitude. We have shown the possibility to employ Au nanocrescents in the detection of haemoglobin, reaching a detection limit of 100 pM. Au/PDMS plasmonic substrates were employed for first measurements on mitochondria, suggesting the possibility to detect via SERS the properties of cytochrome c molecules contained in these organelle. Subsequently we faced the problems related to the in-liquid SERS detection of biomolecules. We have exploited a strategy (LIQUISOR) based on optical forces to push and form SERS-active aggregates of gold nanorods (NR) mixed with proteins. Working on Bovine Serum Albumin (BSA), we have reached a limit of detection of 50nM, we have studied the growth kinetics of the optically induced aggregate, its morphology by SEM images and demonstrated that LIQUISOR can provide quantitative information on the protein concentration. A model has been developed that describes the process of nanorod/protein binding and size increase of the bio-nanorod composite, supported by dynamic light scattering measurements. To assess the efficiency and versatility of the LIQUISOR methodology we have applied it to the detection of Lysozyme (Lys), Hemoglobin (Hgb) and Catalase (Cat). Detection at physiological pH was demonstrated in all cases, reaching sensitivities of few ng/mL (picomolar) and high SERS gains (seven orders of magnitude for Hgb). Finally, we carried out first proof of principle experiments of of high sensitive and selective LIQUISOR detection of MnSOD (4.5 nM) and Ochratoxin A (1 µM), exploiting aptamers to functionalize the gold NRs and add specificity to the LIQUISOR methodology. In the last part of this thesis the attention was moved to Tip enhanced Raman spectroscopy. Firstly we have developed a fast and efficient double-step electrochemical etching for the fabrication of nanotips featuring radius of curvature lower than 35 nm starting from Au wires of 125 µm diameter. Homemade tips were used to obtain TERS spectra of Rhodamine 6G (R6G), Methylene Blue, Crystal violet (CV) and Alizarin. TERS enhancement factors EF higher than four orders of magnitude were obtained. TERS imaging was demonstrated on a molecular film of R6G and CV adsorbed on a flat gold substrate to discriminate aggregates of molecules with a spatial resolution of 3 nm. TERS was finally applied in the biomolecular field to high sensitivity spectroscopy of HypF-N oligomers, analogues of amyloid oligomers, the constitutive elements of amyloid fibrils, responsible for several neurodegenerative diseases. Our first results highlight the potentiality of TERS effect for the detection of small biological systems. Notably, our results evidence the possibility to discriminate among different structural conformation of the oligomers, one of which exhibiting a toxic behavior that leads to the formation of misfolded amyloid fibrils in Parkinson’s disease. Several possible developments are envisaged for both the LIQUISOR and the TERS methodologies. The LIQUISOR is of rapid use (few minutes), experimentally simple (standard micro-spectrometers and commercial nanorods are used), reliable and intrinsically scalable to lab-on-chip devices. Higher specificity can potentially be achieved by centrifuging functionalized NR mixed to the protein in order to separate free molecules in complex fluids from the target proteins interacting with aptamer functionalized NR or employing functionalized surfaces (instead of glass slides) to increase the affinity between BIO-NRCs and substrates and thus to speed up the aggregation process. On the other side higher sensitivity can be potentially achieved adopting silver nanoplatelets, as well as core-shell nanostructures. The use of laser beams in the optical transparency window of biological tissues could enable the application of our scheme in combination with optical injection of nanoparticles into living cells for in-vivo SERS biomolecular detection. TERS, with its high sensitivity and spatial resolution has already demonstrated potentialities in the field of DNA sequencing and can have unique applications in proteomics, allowing for the detection of single amino-acid alterations, e.g. phosphorylation, in complex proteins. The fabrication of new tips, more efficient, less expensive and more reproducible is another future challenge. Besides applications in the nanomedicine field, TERS can have important applications in cultural heritage field, for identification of inks on paper, dyes on statues which can be essential for dating, restoring and conserving the artwork.