Covalent chemistry enables extracellular vesicle purification on nanosubstrates ? toward early detection of hepatocellular carcinoma

A method of assaying for a cancer in a subject comprising:

• selectively capturing an extracellular vesicle (EV) from a sample from said subject, said selectively capturing an EV comprising:
• functionalizing a capture agent for said EV with a first molecule from a first bioorthogonal functional group such that said capture agent remains attachable to said EV and said first molecule is also able to bond to a second molecule from a second bioorthogonal functional group, said second molecule being complementary to said first molecule;
• mixing said functionalized capture agent with said sample such that said functionalized capture agent binds to said EV and such an activated sample is formed;
• functionalizing a capture surface with said second molecule;
• and
• depositing at least a portion of said activated sample on at least a portion of said functionalized capture surface to thereby selectively capture said EV by binding of said molecule with said first molecule;
• releasing said EV from said capture surface;
• assaying a nucleic acid sequence from said EV;
• and
• determining from said assaying of said nucleic acid sequence from said EV whether said cancer is present in said subject,
• wherein said first molecule from said first bioorthogonal functional group and said capture agent are present in a molar ratio of between 2:1 to 10:1.

A method of assaying for a cancer in a subject comprising:

• selectively capturing a plurality of extracellular vesicles (EVs) from a sample from said subject, wherein each extracellular vesicle (EV) of said plurality of EVs is selectively captured comprising:
• functionalizing a capture agent for an EV of said plurality of EVs with a first molecule from a first bioorthogonal functional group such that said capture agent remains attachable to said EV and said first molecule is also able to bond to a second molecule from a second bioorthogonal functional group, said second molecule being complementary to said first molecule;
• mixing said functionalized capture agent with said sample such that said functionalized capture agent binds to said EV and such an activated sample is formed;
• functionalizing a capture surface with said second molecule;
• and
• depositing at least a portion of said activated sample on at least a portion of said functionalized capture surface to thereby selectively capture said EV by binding of said molecule with said first molecule;
• releasing said plurality of EVs from said capture surface;
• assaying a plurality of nucleic acid sequences from said plurality of EVs;
• creating an expression profile of said plurality of nucleic acid sequences, said expression profile comprising a quantification of each of said plurality of nucleic acid sequences;
• comparing said expression profile with a control;
• and
• determining from said comparing of said expression profile with said control whether said cancer is present in said subject,
• wherein said first molecule from said first bioorthogonal functional group and said capture agent are present in a molar ratio of between 2:1 to 10:1.

A kit for selectively capturing an extracellular vesicle (EV) from a sample, comprising:

• a capture agent comprising a first molecule from a first bioorthogonal functional group;
• a substrate comprising a functionalized capture surface having a second molecule from a second bioorthogonal functional group, said second molecule being complementary to said first molecule;
• a cleaving agent;
• instructions for mixing said capture agent with said sample such that said capture agent binds to said EV and such that an activated sample is formed;
• instructions for depositing at least a portion of said activated sample on at least a portion of said functionalized capture surface to thereby selectively capture said EV by binding of said second molecule with said first molecule;
• and
• instructions for using said cleaving agent to release said EV from said capture surface,
• wherein said first molecule from said first bioorthogonal functional group and said capture agent are present in a molar ratio of between 2:1 to 10:1.