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Abstract Background Extracellular Vesicles (EVs) are sub-micrometer lipid-bound particles released by most cell types. They are considered a promising source of cancer biomarkers for liquid biopsy and personalized medicine due to their specific molecular cargo, which provides biochemical information on the state of parent cells. Despite this potential, EVs translation process in the diagnostic practice is still at its birth, and the development of novel medical devices for their detection and characterization is highly required. Results In this study, we demonstrate mid-infrared plasmonic nanoantenna arrays designed to detect, in the liquid and dry phase, the specific vibrational absorption signal of EVs simultaneously with the unspecific refractive index sensing signal. For this purpose, EVs are immobilized on the gold nanoantenna surface by immunocapture, allowing us to select specific EV sub-populations and get rid of contaminants. A wet sample-handling technique relying on hydrophobicity contrast enables effortless reflectance measurements with a Fourier-transform infrared (FTIR) spectro-microscope in the wavelength range between 10 and 3 µm. In a proof-of-principle experiment carried out on EVs released from human colorectal adenocarcinoma (CRC) cells, the protein absorption bands (amide-I and amide-II between 5.9 and 6.4 µm) increase sharply within minutes when the EV solution is introduced in the fluidic chamber, indicating sensitivity to the EV proteins. A refractive index sensing curve is simultaneously provided by our sensor in the form of the redshift of a sharp spectral edge at wavelengths around 5 µm, where no vibrational absorption of organic molecules takes place: this permits to extract of the dynamics of EV capture by antibodies from the overall molecular layer deposition dynamics, which is typically measured by commercial surface plasmon resonance sensors. Additionally, the described metasurface is exploited to compare the spectral response of EVs derived from cancer cells with increasing invasiveness and metastatic potential, suggesting that the average secondary structure content in EVs can be correlated with cell malignancy. Conclusions Thanks to the high protein sensitivity and the possibility to work with small sample volumes—two key features for ultrasensitive detection of extracellular vesicles- our lab-on-chip can positively impact the development of novel laboratory medicine methods for the molecular characterization of EVs. Graphical Abstract

Extracellular vesicles are membrane-delimited structures, involved in several inter-cellular communication processes, both physiological and pathological, since they deliver complex biological cargo. Extracellular vesicles have been identified as possible biomarkers of several pathological diseases; thus, their characterization is fundamental in order to gain a deep understanding of their function and of the related processes. Traditional approaches for the characterization of the molecular content of the vesicles require a large quantity of sample, thereby providing an average molecular profile, while their heterogeneity is typically probed by non-optical microscopies that, however, lack the chemical sensitivity to provide information of the molecular cargo. Here, we perform a study of individual microvesicles, a subclass of extracellular vesicles generated by the outward budding of the plasma membrane, released by two cultures of glial cells under different stimuli, by applying a state-of-the-art infrared nanospectroscopy technique based on the coupling of an atomic force microscope and a pulsed laser, which combines the label-free chemical sensitivity of infrared spectroscopy with the nanometric resolution of atomic force microscopy. By correlating topographic, mechanical and spectroscopic information of individual microvesicles, we identified two main populations in both families of vesicles released by the two cell cultures. Subtle differences in terms of nucleic acid content among the two families of vesicles have been found by performing a fitting procedure of the main nucleic acid vibrational peaks in the 1000–1250 cm−1 frequency range.

We describe the activation of out-of-equilibrium collective oscillations of a macromolecule as a classical phonon condensation phenomenon. If a macromolecule is modeled as an open system—that is, it is subjected to an external energy supply and is in contact with a thermal bath to dissipate the excess energy—the internal nonlinear couplings among the normal modes make the system undergo a nonequilibrium phase transition when the energy input rate exceeds a threshold value. This transition takes place between a state where the energy is incoherently distributed among the normal modes and a state where the input energy is channeled into the lowest-frequency mode entailing a coherent oscillation of the entire molecule. The model put forward in the present work is derived as the classical counterpart of a quantum model proposed a long time ago by Fröhlich in an attempt to explain the huge speed of enzymatic reactions. We show that such a phenomenon is actually possible. Two different and complementary THz near-field spectroscopic techniques—a plasmonic rectenna and a microwire near-field probe—have been used in two different labs to eliminate artifacts. By considering an aqueous solution of a model protein, the bovine serum albumin, we find that this protein displays a remarkable absorption feature around 0.314 THz, when driven in a stationary out-of-thermal equilibrium state by means of optical pumping. The experimental outcomes are in very good qualitative agreement with the theory developed in the first part of the paper and in excellent quantitative agreement with the theoretical result, allowing us to identify the observed spectral feature with a collective oscillation of the entire molecule.