Gerardo Palazzo

Characterization of Covalently Bound Anti-Human Immunoglobulins on Self-Assembled Monolayer Modified Gold Electrodes

Bioconjugated gold surfaces constitute interesting platforms for biosensing applications. The immobilization of antibodies such as anti‐immunoglobulin G and M (anti‐IgG and anti‐IgM) on gold electrodes via self‐assembled monolayers (SAMs) is here studied as a model system for further immunoassays development. The biolayer is characterized by means of X‐ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), a dedicated thin‐film transistor (TFT)‐based platform and electrochemical surface plasmon resonance (EC‐SPR). XPS analysis confirms the presence of all the chemical species involved in the fabrication process as well as the covalent attachment of the antibodies with high reproducibility. Visualization of the biolayer topography by AFM shows nanostructures with a thickness consistent with the actual size of the protein, which is also verified by SPR measurements. EC‐SPR allows taking advantage of complementary electrochemical and optical signals during the functionalization steps. Moreover, the functionalization of gold leads to a change in the work function, which is demonstrated in an electrolyte gated thin‐film transistor configuration. Such configuration enables also to evaluate the electrostatic changes occurring on the gate that are connected with the threshold voltage shifts. The data support that functional biomodified gold surfaces can be reproducibly prepared, which is a prerequisite for further biosensor development.

Counting of peripheral extracellular vesicles in Multiple Sclerosis patients by an improved nanoplasmonic assay and dynamic light scattering

Extracellular vesicles (EVs) are vesicles naturally secreted by the majority of human cells. Being composed by a closed phospholipid bilayer secluding proteins and RNAs they are used to transfer molecular information to other cells, thereby influencing the recipient cell functions. Despite the increasingly recognized relevance of EVs, the clarification of their physiological role is hampered by the lack of suitable analytical tools for their quantification and characterization. In this study, we have implemented a nanoplasmonic assay, previously proposed for the purity of the EV fractions, to achieve a robust analytical protocol in order to quantify the total phospholipid concentration (CPL) and the EVs number. We show how the coupling of the nanoplasmonic assay with serial dilutions of the unknown sample allows, by simple visual inspection, to detect deviations from the physiological EVs content. The use of a response that depends on the absorbance values at three wavelengths permits to reduce the limit of detection of CPL to 5u202fμM (total) and the limit of quantification to 35u202fμM. We also propose a method that takes into account the spread in EV size when the concentration of phospholipids is turned into a concentration of vesicles. The proposed analytical protocol is successfully applied to a small cohort of Multiple Sclerosis patients examined in different stages of their clinical diseases.