Method of determining a protein in a sample

1. A method of determining a protein in a sample, comprising the steps of:

(a) providing an aptamer, which is functionalized at the 5′-end with: i) a labeling reagent comprising a photo-activatable crosslinking moiety; ii) a labeling moiety selected from the group consisting of biotin and desthiobiotin, and iii) a linker moiety connecting the labeling reagent to the 5′-end of the aptamer;

(b) incubating a sample comprising an aptamer target protein with the functionalized aptamer of step (a);

(c) irradiating the sample with ultraviolet light for crosslinking the aptamer via the photo-activatable crosslinking moiety to the protein, wherein the aptamer is not cleaved by the irradiation; and

(d) determining the protein via the labeling moiety.

2. The method according to claim 1 , wherein the photo-activatable crosslinking moiety is selected from the group comprising aryl azides (phenyl azide), diazirine derivatives such as 3-trifluoromethyl-3-phenyl-diazirine (TPD), and benzophenone derivatives.

3. The method according to claim 1 , wherein the linker moiety provides a spacer arm length of about 6 Å to 45 Å between the 5′-end of the aptamer and the crosslinking moiety.

4. The method according to claim 1 , wherein the labeling reagent is Sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate.

5. The method according to claim 1 , wherein the method comprises incubating a biotin-labeled cross-linked aptamer-protein complex formed in step (c) with avidin-, neutravidin-, or streptavidin-coated magnetic beads.

6. The method according to claim 1 , wherein step (d) comprises determining a protein in a cell, or in an exosome, or in a circulating microvesicle.

7. The method according to claim 1 , wherein step (d) includes determining a protein on the surface of a cell, or an exosome, or a circulating microvesicle.

8. The method according to claim 5 , further comprising sorting of the magnetic beads.

9. The method of claim 3 , wherein the spacer arm length is about 20 Å to 40 Å.

10. The method of claim 1 , wherein the aptamer is functionalized at the 5′-end with a construct consisting of a photo-activatable crosslinking moiety, a labeling moiety selected from the group consisting of biotin and desthiobiotin, and a linker moiety connecting the labeling reagent to the 5′-end of the aptamer.

11. The method of claim 1 , wherein the aptamer is functionalized at the 5′-end with a construct consisting of the following structure: