Introduction: Surface Enhanced Raman spectroscopy (SERS) promises to be a powerful resource to provide information about the biochemical content of Extracellular Vesicles (EVs) in a fast and reproducible way. We firstly explored the ability of plasmonic and non-plasmonic SERS to probe nanosized EV populations separated from human serum of patients affected by Multiple Myeloma (MM) or Parkinsos’s disease (PD) and from healthy (H) donors. Typically, metal nanoparticle (NP) with a plasmonic resonance (e.g. Au) are utilized to enhance the Raman response (plasmonic SERS). However excited plasmonic NPs generate local heating and energy release, thereby inducing instability and low reproducibility, especially with organic or biological analytes. For this reason we also considered to probe EVs with innovative T-rex beads made of SiO2/TiO2 core/shell colloids that enhance the Raman fingerprint of the analyte by non-plasmonic SERS, thus expected to show a lower ability impact on the stability of the adsorbed EVs. Methods: EVs from serum of H patients, with MM or PD were purified using sequential centrifugation steps and discontinuous sucrose gradients. Samples were biochemically characterized by Western Blot analysis. Positive fractions to typical exosomal markers were pooled and further characterized for biophysical characteristics by atomic force microscopy (AFM), colloidal nanoplasmonic assays, and an agarose gel. EVs were then targeted with 15 nm Au NPs and analyzed by conventional SERS. In alternative EVs were coupled with T-rex beads for non-plasmonic SERS . Results: The colloidal nanoplasmonic assay allowed us to assess purity and determine the molar concentration of the EV formulations. AFM imaging confirmed the formulation to be composed of nanosized EV populations (50-100 nm). Both plasmonic and non-plasmonic SERS experiments gave promising results in terms of the possibility to use SERS profiling to identify each of the H, MM and PD EV populations. Our contribution will focus on presenting and discussing the last updates of these results (further experiments are ongoing). The institutional review board of Azienda Ospedaliera Spedali Civili of Brescia approved the study in adherence with the Declaration of Helsinki. This project was financed by the BIOMANE grant from the University of Brescia 2015.

Abstract Book: ISEV2017

Paolini Lucia;BONTEMPI, NICOLO';Radeghieri Annalisa;CASTELLI, ANNA;ZENDRINI, ANDREA;BUSATTO, SARA;Monti Eugenio;Alessandri Ivano;Bergese Paolo.
2017

Abstract

Introduction: Surface Enhanced Raman spectroscopy (SERS) promises to be a powerful resource to provide information about the biochemical content of Extracellular Vesicles (EVs) in a fast and reproducible way. We firstly explored the ability of plasmonic and non-plasmonic SERS to probe nanosized EV populations separated from human serum of patients affected by Multiple Myeloma (MM) or Parkinsos’s disease (PD) and from healthy (H) donors. Typically, metal nanoparticle (NP) with a plasmonic resonance (e.g. Au) are utilized to enhance the Raman response (plasmonic SERS). However excited plasmonic NPs generate local heating and energy release, thereby inducing instability and low reproducibility, especially with organic or biological analytes. For this reason we also considered to probe EVs with innovative T-rex beads made of SiO2/TiO2 core/shell colloids that enhance the Raman fingerprint of the analyte by non-plasmonic SERS, thus expected to show a lower ability impact on the stability of the adsorbed EVs. Methods: EVs from serum of H patients, with MM or PD were purified using sequential centrifugation steps and discontinuous sucrose gradients. Samples were biochemically characterized by Western Blot analysis. Positive fractions to typical exosomal markers were pooled and further characterized for biophysical characteristics by atomic force microscopy (AFM), colloidal nanoplasmonic assays, and an agarose gel. EVs were then targeted with 15 nm Au NPs and analyzed by conventional SERS. In alternative EVs were coupled with T-rex beads for non-plasmonic SERS . Results: The colloidal nanoplasmonic assay allowed us to assess purity and determine the molar concentration of the EV formulations. AFM imaging confirmed the formulation to be composed of nanosized EV populations (50-100 nm). Both plasmonic and non-plasmonic SERS experiments gave promising results in terms of the possibility to use SERS profiling to identify each of the H, MM and PD EV populations. Our contribution will focus on presenting and discussing the last updates of these results (further experiments are ongoing). The institutional review board of Azienda Ospedaliera Spedali Civili of Brescia approved the study in adherence with the Declaration of Helsinki. This project was financed by the BIOMANE grant from the University of Brescia 2015.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11379/502589
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