RAFFAELLA POLITO

PhD

PhD program:: XXXIV


tutor: Paolo Postorino
advisor: Leonetta Baldassarre

Thesis title: Plasmonic and photonic strategies to measure conformational changes of transmembrane proteins at the nanoscale

The ability of identifying chemically a single bio macro-molecule, also assessing its three dimensional conformation, would be of great interest in several fields as chemistry, environmental science and biology. Mid-Infrared spectroscopy (mid-IR; λ ~ 2-20 μm) would be the label-free technique of choice in this sense, as it provides information on the specific chemical bonds of the molecules together with their three dimensional arrangement. Unfortunately, the diffraction limit and the very small absorption cross section of IR radiation limit the use of mid-IR spectroscopy to large number of nominally identical proteins. To increase the sensitivity of the mid-IR spectroscopy, several strategies have been employed aimed at confining and enhancing the electromagnetic field into sub-wavelength volumes. In this thesis, I tackle the issue of decreasing the number of probed molecules in the mid-IR with two different approaches. A “plasmonic approach” has been used to obtain a highly enhanced electric field in the nanogap between a gold-coated tip of an Atomic Force Microscope (AFM) and a gold surface allowing the study of functional conformational changes of proteins at the nanoscale. On the other hand, a “photonic approach” has been used to produce highly confined surface states sustained by an one-dimensional photonic crystal for prospective easy-to-use sensing devices of single molecular layers. I leveraged on a nanospectroscopic technique to investigate subtle and reversible light-induced conformational changes of transmembrane proteins embedded in individual nanometre-tick cell membrane patches. The used experimental nanospectroscopy platform is based on the coupling of a tunable mid-IR quantum cascade laser and an AFM. The sensitivity to individual cell membrane patches is achieved by exploiting a plasmonic field enhancement in the nanogap between the gold-coated AFM tip and a gold surface used as a sample support. Moreover, a difference spectroscopy scheme was developed in order to have a high-enough signal-to-noise ratio to isolate relative absorption variations smaller than 10−2. Noteworthy, my results demonstrate that the light-induced functional activity of these proteins is retained at the single membrane layer, when the proteins are in contact with metallic surfaces. The work presented thus opens the way towards the study of these transmembrane proteins in their inherently heterogeneous native membranes. In order to probe routinely conformational changes of a single protein monolayer, however, one should develop a different strategy that can be used to confine and enhance the electromagnetic field in sub-wavelength volumes, comparable to the biomolecular scale, similarly to what is done in attenuated total reflection or sensors based on surface electromagnetic waves. Instead of exploiting Surface Plasmon Polaritons (SPPs) in the mid-IR, that suffer for a poor confinement due to the long wavelengths employed, I have studied the Bloch Surface Waves (BSWs) sustained by an one-dimensional photonic crystal in the mid-IR range. BSWs have found application in the visible and near-IR range as low-loss alternatives to SPPs in biosensors. In this thesis I have translated this concept to the mid-IR, reporting on a photonic crystal consisting of a periodic structure of two ZnS/CaF2 pairs. Mid-IR BSWs biosensors show several advantages with respect to those based on SPPs as they give rise to higher field confinement at the wavelength of our interest (the amide I absorption at 6 µm) and can sustain both transverse-electric and transverse-magnetic polarizations at the surface. This would be extremely relevant for the investigation of conformational changes of proteins attached to the surface itself, making mid-IR BSWs biosensor forefront runners for label-free and polarization-dependent sensing devices.

Research products

  • 11573/1274683 - 2019 - Tip-Enhanced Infrared Difference-Nanospectroscopy of the Proton Pump Activity of Bacteriorhodopsin in Single Purple Membrane Patches (01a Articolo in rivista)
    GILIBERTI, VALERIA; POLITO, RAFFAELLA; SCHADE, ULRICH BRUNO; ZANETTI POLZI, LAURA; DAIDONE, ISABELLA; BALDASSARRE, LEONETTA; ORTOLANI, MICHELE
  • 11573/1410219 - 2019 - Spectroscopic Evidence of Bloch Surface Waves in the Mid Infrared (04d Abstract in atti di convegno)
    ORTOLANI, MICHELE; OCCHICONE, AGOSTINO; GILIBERTI, VALERIA; SINIBALDI, ALBERTO; POLITO, RAFFAELLA; NUCARA, ALESSANDRO; BALDASSARRE, LEONETTA; MICHELOTTI, FRANCESCO
  • 11573/1410267 - 2018 - Light-induced conformational changes of protein receptors probed by difference mid-IR microspectroscopy (04d Abstract in atti di convegno)
    POLITO, RAFFAELLA; NUCARA, ALESSANDRO; CALVANI, PAOLO; BALDASSARRE, LEONETTA; ORTOLANI, MICHELE
  • 11573/1410257 - 2019 - Difference mid-IR nanospectroscopy on individual patches of purple membranes: The proton pump activity of bacteriorhodopsin at the nanoscale (04d Abstract in atti di convegno)
    POLITO, RAFFAELLA; BALDASSARRE, LEONETTA; ORTOLANI, MICHELE
  • 11573/1500060 - 2021 - Spectral characterization of mid-infrared Bloch Surface Waves excited on a truncated 1D photonic crystal (01a Articolo in rivista)
    OCCHICONE, AGOSTINO; POLITO, RAFFAELLA; GILIBERTI, VALERIA; SINIBALDI, ALBERTO; NUCARA, ALESSANDRO; MICHELOTTI, FRANCESCO; ORTOLANI, MICHELE; BALDASSARRE, LEONETTA
  • 11573/1646082 - 2020 - Plasmonic Superchiral Lattice Resonances in the Mid-Infrared (01a Articolo in rivista)
    POLITO, RAFFAELLA; BALDASSARRE, LEONETTA; NUCARA, ALESSANDRO; ORTOLANI, MICHELE
  • 11573/1657462 - 2021 - Infrared nanospectroscopy study of the light-induced conformational changes of Channelrhodopsin (04d Abstract in atti di convegno)
    TEMPERINI, MARIA ELEONORA; POLITO, RAFFAELLA; INTZE, ANTONIA; SCHADE, ULRICH BRUNO; BALDASSARRE, LEONETTA; ORTOLANI, MICHELE; GILIBERTI, VALERIA
  • 11573/1654895 - 2022 - Low-Temperature Stability and Sensing Performance of Mid-Infrared Bloch Surface Waves on a One-Dimensional Photonic Crystal (01a Articolo in rivista)
    OCCHICONE, AGOSTINO; POLITO, RAFFAELLA; MICHELOTTI, FRANCESCO; ORTOLANI, MICHELE; BALDASSARRE, LEONETTA; SINIBALDI, ALBERTO; CALVANI, PAOLO; NUCARA, ALESSANDRO
  • 11573/1560699 - 2020 - Infrared nanospectroscopy and nanoimaging of individual cell membranes and microvesicles exposed to air (01a Articolo in rivista)
    TEMPERINI, MARIA ELEONORA; GILIBERTI, VALERIA; POLITO, RAFFAELLA; BALDASSARRE, LEONETTA
  • 11573/1656419 - 2021 - Conformational changes of a membrane protein determined by infrared difference spectroscopy beyond the diffraction limit (01a Articolo in rivista)
    POLITO, RAFFAELLA; TEMPERINI, MARIA ELEONORA; SCHADE, ULRICH BRUNO; BALDASSARRE, LEONETTA; GILIBERTI, VALERIA
  • 11573/1656418 - 2021 - Infrared Nanospectroscopy of Individual Extracellular Microvesicles (01a Articolo in rivista)
    POLITO, RAFFAELLA; TEMPERINI, MARIA ELEONORA; ORTOLANI, MICHELE; BALDASSARRE, LEONETTA; GILIBERTI, VALERIA
  • 11573/1657460 - 2021 - Conformational changes of an oriented film of photosensitive proteins observed by polarized ATR infrared spectroscopy (04d Abstract in atti di convegno)
    TEMPERINI, MARIA ELEONORA; POLITO, RAFFAELLA; INTZE, ANTONIA; BALDASSARRE, LEONETTA; GILIBERTI, VALERIA
  • 11573/1657463 - 2022 - Mid-infrared nanospectroscopy of individual DNA-binding protein oligomers (04d Abstract in atti di convegno)
    INTZE, ANTONIA; TEMPERINI, MARIA ELEONORA; POLITO, RAFFAELLA; TARTAGLIA, GIAN GAETANO; GILIBERTI, VALERIA
  • 11573/1657464 - 2022 - A Quantum Cascade Laser Microscope for Time-resolved Study of Membrane Proteins (04d Abstract in atti di convegno)
    TEMPERINI, MARIA ELEONORA; POLITO, RAFFAELLA; INTZE, ANTONIA; GILLIBERT, RAYMOND; BALDASSARRE, LEONETTA; GILIBERTI, VALERIA

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