Extracellular vesicle characterization systems

1 . A method of detecting binding sites on an extracellular vesicle, comprising:

obtaining a liquid sample containing extracellular vesicles;

attaching reporters to binding sites on the extracellular vesicles; and

optically interrogating the reporters to characterize a number of bindings of the reporters to the extracellular vesicles; and

wherein attaching reporters to binding sites on the extracellular vesicles comprises:

providing a detection system comprising a binding site recognition element linked to biotin and a biotin-binding protein attached to the reporter; and

allowing the liquid sample containing the extracellular vesicles to interact with the detection system.

2 . The method of claim 1 further comprising treating the liquid sample to amplify the optical response.

3 . The method of claim 2 wherein treating the liquid sample to amplify the optical response comprises:

attaching nanoparticles to the extracellular vesicles, wherein the nanoparticles are functionalised with biotin molecules;

allowing the liquid sample containing the extracellular vesicles to interact with the detection system and the nanoparticles such that one biotin-binding protein molecule links the antibody linked to biotin with one of the nanoparticles functionalised with biotin molecules which in turn links to multiple biotin-binding protein molecules each attached to a respective reporter.

4 . The method of claim 3 wherein an average dimension of the nanoparticles is smaller than an average dimension of the extracellular vesicles.

5 . The method of claim 1 , further comprising attaching the extracellular vesicles to magnetic beads.

6 . The method of claim 5 , wherein magnetic beads are functionalised with one or more of surface-bound binding agents, antibodies, a photo-cleavable linker, a disulfide bridge, DNA, RNA, or DNA-RNA hybrid.

7 . The method of claim 1 , wherein attaching reporters to binding sites comprises:

attaching a first reporter to a first binding site; and

attaching a second reporter to a second binding site, wherein the first and second reports exhibit a different optical response, and optionally wherein the different optical response comprises the first and second reporters providing optical responses at different wavelengths.

8 . The method of claim 7 , wherein the detection system comprises a second binding site recognition element linked to biotin and a biotin-binding protein attached to the second reporter, wherein the second binding site recognition element is different to the first binding site recognition element.

9 . The method of claim 1 , wherein attaching reporters to binding sites on the extracellular vesicles comprises:

attaching nanoparticles to the extracellular vesicles, wherein an average dimension of the nanoparticles is smaller than an average dimension of the extracellular vesicles, and

attaching the reporters to the nanoparticles.

10 . The method of claim 1 , wherein the reporter comprises either:

(i) an enzyme, wherein the comprises horseradish peroxidase (HRP), or

(ii) a fluorophore, wherein the fluorophore comprises Alexa Fluor 488.

11 . The method of claim 1 , wherein the binding site recognition element is an antibody, aptamer, or lipid-binding protein.

12 . The method of claim 1 , wherein the binding sites comprise an epitope, membrane lipid or a membrane bound protein.

13 . The method of claim 1 , further comprising determining, based on the number of bindings of the reporters, at least one of:

a concentration of the reporters;

a concentration of extracellular vesicles;

the concentration of extracellular vesicle proteins; or

the concentration of extracellular vesicle lipids.

14 . The method of claim 1 , wherein the extracellular vesicle comprises one or more surface markers, optionally wherein the one or more surface markers comprise tetraspanin, and further optionally wherein the tetraspanin comprises one or more of CD9, CD63, CD81, CD326, CD82, CD37 or CD41.

15 . The method of claim 1 , wherein the reporters comprise enzymatic, chemiluminescent, or fluorescent reporters, and wherein optically interrogating the reporters comprises measuring an optical response of the liquid sample.

16 . The method of claim 1 , further comprising, prior to optically interrogating the reporters, treating the reporters with a reagent.

17 . The method of claim 1 , wherein optically interrogating the reporters comprises measuring an optical characteristic of the reporters, wherein the optical characteristic is an absorbance, a transmittance, a reflectance or an intensity of fluorescence or chemiluminescence.

18 . The method of claim 1 , further comprising determining, based on the number of bindings of the reporters, an indication of at least one of a liver disease, breast cancer or a neurological disease, wherein the neurological disease is Alzheimer's disease.

19 . A method of determining a number of binding sites on an extracellular vesicle, comprising:

obtaining a liquid sample containing extracellular vesicles;

attaching electrically conducting nanoparticles to the extracellular vesicles;

attaching reporters to binding sites on the extracellular vesicles;

interrogating the reporters to determine a total number of bindings of the reporters to the extracellular vesicles;

determining a number of extracellular vesicles in the liquid sample by sensing an electrical response of the liquid sample using a pair of electrodes separated by less than an average maximum dimension of the electrically conducting nanoparticles; and

combining the determined total number of bindings and the determined number of extracellular vesicles in the liquid sample to determine a number of binding sites per extracellular vesicle.

20 . The method as claimed in claim 19 further comprising limiting a number of electrically conducting nanoparticles attached to each extracellular vesicle by:

selectively attaching the extracellular vesicles to magnetic beads in the liquid sample, wherein the magnetic beads have an average maximum dimension which is larger than the average maximum dimension of the electrically conducting nanoparticles;

processing the liquid sample to select magnetic beads with extracellular vesicles attached to obtain the liquid sample containing extracellular vesicles;

attaching the electrically conducting nanoparticles to the extracellular vesicles whilst attached to the magnetic beads; and

detaching the magnetic beads from the selected extracellular vesicles before sensing the electrical response.

21 . The method as claimed in claim 19 wherein an average dimension of the conducting nanoparticles is larger than an average dimension of the extracellular vesicles.

22 . The method as claimed in claim 19 , wherein attaching conducting nanoparticles to the extracellular vesicles comprises:

obtaining conducting nanoparticles with a biotin-binding protein and a first binding site recognition element linked to biotin, and linked by the biotin to the biotin-binding protein; and

allowing the liquid sample containing the extracellular vesicles to interact with the conducting nanoparticles.

23 . The method as claimed in claim 19 , wherein attaching reporters to binding sites on the extracellular vesicles comprises at least one of:

(i) providing a detection system comprising a second binding site recognition element linked to biotin and a biotin-binding protein attached to the reporter; and

allowing the liquid sample containing the extracellular vesicles to interact with the detection system;

(ii) attaching first reporters to first binding sites on the extracellular vesicles and second reporters to second binding sites on the extracellular vesicles, wherein interrogating the reporters to determine a total number of bindings of the reporters to the extracellular vesicles comprises interrogating the first reporters to determine a total number of bindings of the first reporters and interrogating the second reporters to determine a total number of bindings of the second reporters, and wherein the method comprises determining a number of binding sites per extracellular vesicle for each of the first binding sites and the second binding sites; and

iii) attaching smaller nanoparticles to the extracellular vesicles, wherein the smaller nanoparticles have an average dimension smaller than an average dimension of the extracellular vesicles, and attaching the reporters to the smaller nanoparticles.

24 . The method as claimed in claim 19 , wherein:

attaching conducting nanoparticles to the extracellular vesicles comprises:

obtaining conducting nanoparticles with a biotin-binding protein and a first binding site recognition element linked to biotin, and linked by the biotin to the biotin-binding protein; and

allowing the liquid sample containing the extracellular vesicles to interact with the conducting nanoparticles; and

attaching reporters to binding sites on the extracellular vesicles comprises:

providing a detection system comprising a second binding site recognition element linked to biotin and a biotin-binding protein attached to the reporter; and

allowing the liquid sample containing the extracellular vesicles to interact with the detection system;

the method comprising attaching the conducting nanoparticles then attaching the reporters.

25 . The method as claimed in claim 19 , wherein sensing the electrical response of the liquid sample comprises at least one of:

(i) measuring an electrical current flowing between the electrodes; and

(ii) concentrating the electrically conducting nanoparticles in a vicinity of the electrodes using dielectrophoresis.

26 . The method as claimed in claim 19 , wherein the reporters comprise enzymatic, chemiluminescent, or fluorescent reporters, and wherein interrogating the reporters comprises measuring an optical response of the liquid sample.

27 . The method as claimed in claim 19 , further comprising detaching the extracellular vesicles from the electrically conducting nanoparticles and/or from the reporters, separating the extracellular vesicles from the electrically conducting nanoparticles, and characterizing contents of the extracellular vesicles.

28 . The method as claimed in claim 19 , wherein the extracellular vesicles are exosomes and the binding sites are epitopes.

29 . The method of detecting a disease in a biofluid sample from a patient using the method of claim 19 , comprising obtaining the liquid sample containing extracellular vesicles from the biofluid sample.

30 . A system for detecting binding sites on an extracellular vesicle, wherein the system is configured to:

accept a liquid sample containing extracellular vesicles;

attach reporters to binding sites on the extracellular vesicles; and

optically interrogate the reporters to characterize a number of bindings of the reporters to the extracellular vesicles; and

wherein attaching reporters to binding sites on the extracellular vesicles comprises:

providing a detection system comprising a binding site recognition element linked to biotin and a biotin-binding protein attached to the reporter; and

allowing the liquid sample containing the extracellular vesicles to interact with the detection system.

31 . The system of claim 30 , comprising one or more optical sensors for optically interrogating the reporters.

32 . The system of claim 30 , wherein the system is configured to attach the extracellular vesicles to magnetic beads.

33 . A system for determining a number of binding sites on an extracellular vesicle, wherein the system is configured to:

accept a liquid sample containing extracellular vesicles;

attach electrically conducting nanoparticles to the extracellular vesicles;

attach reporters to binding sites on the extracellular vesicles;

interrogate the reporters to determine a total number of bindings of the reporters to the extracellular vesicles;

determine a number of extracellular vesicles in the liquid sample by sensing an electrical response of the liquid sample using a pair of electrodes separated by less than an average maximum dimension of the electrically conducting nanoparticles; and

combine the determined total number of bindings and the determined number of extracellular vesicles in the liquid sample to determine number of binding sites per extracellular vesicle.

34 . The system of claim 33 further configured to attach the extracellular vesicles to magnetic beads in the liquid sample before attaching the conducting nanoparticles to the extracellular vesicles.