The invention relates to a method for detecting and/or quantifying extracellular vesicles (EVs) which are present in a biological fluid sample using a solid surface functionalized with a capture molecule, a detection molecule and polycationic polymers. The method of the invention is useful for the detection and/or quantification of biomarkers present in the surface of the EVs as well as in the diagnostic of diseases.
1. A method for the detection and/or quantification of extracellular vesicles (EVs) from a biological fluid sample comprising: a) Obtaining a biological fluid sample of volume between 5 and 45 µL; b) Contacting the biological fluid sample obtained in a) to a solid surface functionalized with at least one capturing molecule capable of binding EVs, obtaining a first solution; c) Incubating the first solution obtained in step b) with at least one detection molecule capable of binding to an epitope in the EVs; and d) Detecting and/or quantifying the detection molecule from step c) bound to the epitope of EVs. 2. The method according to claim 1, wherein a cationic polymer is added to the biological fluid sample obtained in step a). 3. The method according to claims 1 or 2 wherein the biological fluid sample is selected from the list consisting of: urine, serum, plasma, amniotic liquid, cerebrospinal fluid, ascites, bronchoalveolar wash, synovial liquid, breast milk, saliva, bone marrow and blood. 4. The method according to any one of claims 1 to 3 wherein the capturing molecule functionalized to the solid surface and/or the detection molecule is selected from a list consisting of: annexin, lectin and immunoglobulin; wherein preferably the capture molecule and the detection molecule are different. 5. The method according to claim 4 wherein the immunoglobulin is an antibody selected from the list consisting of: anti-CD63, anti-CD81, anti-CD9, anti-tetraspanins, anti-EpCAM and anti-MICA antibody; the annexin is selected from a list consisting of: annexin V, A1, A2, A4 and A5; and the lectin is selected from a list consisting of: Solanum tuberosum lectin, Sambucus nigra agglutinin lectin, Sambucus sieboldiana agglutinin lectin, Trichosanthes japonica agglutinin II lectin, galectin 1 and galectin 3. 6. The method according to any one of claims 2 to 5 wherein the cationic polymer added to the biological fluid sample is selected from the list consisting of: poly-L-lysine, hexadimethrine bromide, gelatin, acrylamide chloride, diallyldimethylammonium chloride, cationic chitosan, cationic cellulose, cationic cyclodextrin, polyethyleneimine, polyamidoamines, polyamino-co-ester, polydimethylaminoethylmethacrylate and cationic dextran, preferably poly-L-lysine and hexadimethrine bromide. 7. The method according to any of claims 1 to 6 wherein the solid surface are microparticles, preferably polymer microparticles, latex microparticles or magnetic microparticles. 8. The method according to any one of claims 1 to 7 wherein the biological fluid sample is obtained from a mammal, preferably a human. 9. The method according to any one of claims 1 to 8 wherein the detection in step d) is made by a laser-based detection instrument or flow cytometry, preferably flow cytometry. 10. The method according to any one of claims 1 to 9 wherein the EVs are exosomes. 11. The method according to any one of claims 1 to 10 wherein the detection molecule of step c) is conjugated to a detectable label. 12. A method for the detection and/or identification of biomarkers of a disease in EVs which comprises the steps of the method according to any one of claims 1 to 11, and an additional step (e) in which disease biomarkers in EVs are phenotyped. 13. An in vitro method for the diagnosis of a disease which comprises the steps of the method according to any one of claims 1 to 11, wherein the capturing molecule capable of binding EVs in step b) binds to a disease biomarker or the epitope in the EV from step c) is an epitope of a disease biomarker and the binding by the capturing molecule to a disease biomarker in the step b) or the binding by the detection molecule to an epitope in the EV from step c) is indicative of disease. 14. The method according to claim 12 or the method according to claim 13 wherein the disease is selected from the list consisting of: cancer, autoimmune diseases, inflammatory diseases, immunodeficiencies, infectious diseases, pathogen originated diseases, cardiovascular diseases, degenerative diseases, hormone disorder related diseases, and metabolic diseases. 15. The method according to claim 14, wherein the disease is cancer, Alzheimer's disease or diabetes.