title: Electrochemical Sandwich Immunosensor for Determination of Exosomes Based on Surface Marker-Mediated Signal Amplification.
abstract: Extracellular vesicles (EVs), namely, exosomes and microvesicles, are important mediators of intercellular communication pathways. Since EVs can be detected in a variety of biofluids and contain a specific set of biomarkers which are reminiscent of their parental cells, they show great promise in clinical diagnostics as EV analysis can be performed in minimally invasive liquid biopsies. However, reliable, fast and cost-effective methods for their determination are still needed, especially if decentralized analysis is intended. In this study, we developed an electrochemical biosensor which works with 1.5 μL sample volume and can detect as low as 200 exosomes per microliter, with a linear range spanning almost 4 orders of magnitude. The sensor is specific and readily differentiates exosomes from microvesicles in samples containing 1000-fold excess of the latter. Capability of detecting exosomes in real samples (diluted serum) was shown. This was achieved by immobilizing rabbit antihuman CD9 antibodies on gold substrates and using monoclonal antibodies against CD9 for detection of captured exosomes. Signal amplification is presumably obtained from the fact that multiple detector antibodies bind to the surface of each captured vesicle. Detection is performed based on electrochemical reduction of 3,3',5,5'-tetramethyl benzidine (TMB) after addition of horseradish peroxidase (HRP)-conjugated anti-IgG antibodies. This amperometric biosensor can be easily incorporated into future miniaturized and semiautomatic devices for EV determination.
authors.affiliation: Analytical Biochemistry Unit, Nuclear Research Center, Faculty of Sciences, Universidad de la República , Mataojo 2055, Montevideo 11400, Uruguay.
authors.forename: Ximena
authors.initials: X
authors.lastname: Doldán
authors.affiliation: Analytical Biochemistry Unit, Nuclear Research Center, Faculty of Sciences, Universidad de la República , Mataojo 2055, Montevideo 11400, Uruguay.
authors.forename: Pablo
authors.initials: P
authors.lastname: Fagúndez
authors.affiliation: Functional Genomics Laboratory, Institut Pasteur de Montevideo . Mataojo 2020, Montevideo 11400, Uruguay.|Department of Medicine, Faculty of Medicine, Universidad de la República , Av. Italia S/N, Montevideo 11600, Uruguay.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Cayota
authors.affiliation: Analytical Biochemistry Unit, Nuclear Research Center, Faculty of Sciences, Universidad de la República , Mataojo 2055, Montevideo 11400, Uruguay.
authors.forename: Justo
authors.initials: J
authors.lastname: Laíz
authors.affiliation: Analytical Biochemistry Unit, Nuclear Research Center, Faculty of Sciences, Universidad de la República , Mataojo 2055, Montevideo 11400, Uruguay.|Functional Genomics Laboratory, Institut Pasteur de Montevideo . Mataojo 2020, Montevideo 11400, Uruguay.
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Tosar
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004566:Electrodes; D004867:Equipment Design; D055354:Exosomes / Q000737:chemistry*; D006046:Gold / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D018796:Immunoconjugates / Q000737:chemistry; D057230:Limit of Detection; D061986:MCF-7 Cells; D011817:Rabbits; D011934:Reagent Strips / Q000032:analysis; D060245:Tetraspanin 29 / Q000032:analysis
chemical_list: D055910:Antibodies, Immobilized; D018796:Immunoconjugates; D011934:Reagent Strips; D060245:Tetraspanin 29; D006046:Gold; D006735:Horseradish Peroxidase