Nanopipette analysis of polymers
The disclosure relates to devices and instruments for detecting and individually analyzing biomolecules, biomolecular complexes and biomolecules with ligands attached thereon.
1. A method for analyzing one or more macromolecules, the method comprising:
i. attaching one or more macromolecules to a fixed location on a solid phase surface;
ii. providing a first electrode in fluidic communication with said one or more macromolecules through an ionic aqueous buffer;
iii. providing a nanopipette containing a second electrode;
iv. applying a potential difference between the first and second electrodes to induce the flow of ions;
v. bringing the nanopipette in sufficient proximity to a macromolecule such that the macromolecule enters into the nanopipette; and
vi. measuring an ion flow.
2. The method of claim 1 , optionally measuring ion flow prior to and after the macromolecule enters the nanopipette.
3. A method according to claim 1 , wherein the macromolecule is a polymer.
4. A method according to claim 3 , wherein the polymer is a nucleic acid or peptide.
5. A method according to claim 4 , wherein the first electrode has a −ve bias and the second electrode has +ve bias.
6. A method according to claim 5 , wherein the nucleic acid is electrophoretically stretched towards the second electrode (+ve bias) due to its negatively charged backbone.
7. A method according to claim 6 , wherein the nanopipette is translated in the Z direction with respect to the surface.
8. A method according to claim 6 , wherein a change in ion flux is measured as the nanopipette is translated in the z direction with respect to the surface.
9. A method according to claim 8 , wherein level of ion flux is correlated with z position.
10. A method according to claim 9 , wherein the nanopipette to surface distance is varied repetitively.
11. A method according to claim 10 , wherein the repeated measurements are used to obtain an average measurement.
12. A method according to claim 11 , wherein the molecule is a nucleic acid and the average measurement is used to make a base call.
13. A method according to claim 11 , wherein the macromolecule is a polypeptide and the average measurement is used to detect an amino acid of the polypeptide.
14. A method according to claim 10 , wherein the macromolecule is a nucleic acid and a base call is made for each repetition.
15. A method according to claim 10 , wherein the macromolecule is a polypeptide and the average measurement is used to detect an amino acid of the polypeptide for each repetition.
16. A method according to claim 1 wherein the macromolecule is released from the nanopipette.
17. A method according to claim 16 , wherein the macromolecule is captured again by the nanopipette.
18. A method according to claim 16 , wherein the solid phase surface is mounted on a stage which is translated in the X or Y direction in relation to the nanopipette or vice versa.
19. A method according to claim 18 , wherein a second macromolecule enters into the nanopipette.
20. A method according to claim 19 , wherein one or more of steps i and vi are repeated for the second macromolecule.
21. A method according to claim 1 , wherein the macromolecule enters the nanopipette through a biological nanopore embedded on an aperture of the nanopipette in sufficient proximity to the macromolecule.
22. A method for analyzing one or more macromolecules selected from the group consisting of nucleic acids and polypeptides, the method comprising:
i. attaching one or more macromolecules to a fixed location on a solid phase surface;
ii. providing a first electrode in fluidic communication with said one or more macromolecules through an ionic aqueous buffer;
iii. providing a nanopipette containing a second electrode;
iv. applying a potential difference between the first and second electrodes to induce the flow of ions;
v. bringing the nanopipette in sufficient proximity to a macromolecule such that the macromolecule enters into the nanopipette; and
vi. measuring an ion flow.