TARGET DETECTION WITH NANOPORE

• US 20140329225A1
• Filed: 05/05/2014
• Published: 11/06/2014
• Est. Priority Date: 05/06/2013
• Status: Abandoned Application

• Current Assignee: Two Pore Guys, Inc.
• Original Assignee: Two Pore Guys, Inc.
• Inventors: Morin, Trevor J.
• Application Number:
  US14/270,283
• Publication Number:
  US 20140329225A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: B81C 1/00158 Diaphragms, membranes manufacture process for semi-permeable inorganic membranes B01D67/0039
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/531 characterised by the capture moiety being a protein for target oligonucleotides
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/4473 by electric means
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68
• CPC Class Codes: G01N 33/5302 Apparatus specially adapted for immunological test procedures
• CPC Class Codes: G01N 33/536 with immune complex formed in liquid phase
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/56911 Bacteria
• CPC Class Codes: G01N 33/56983 Viruses
• CPC Class Codes: G01N 33/574 for cancer

Abstract

Provided are methods for detecting a target molecule or particle suspected to be present in a sample, comprising (a) contacting the sample with (i) a fusion molecule comprising a ligand capable of binding to the target molecule or particle and a binding domain, and (ii) a polymer scaffold comprising at least one binding motif to which the binding domain is capable of binding, under conditions that allow the target molecule or particle to bind to the ligand and the binding domain to bind to the binding motif; (b) loading the polymer into a device comprising a pore that separates an interior space of the device into two volumes, and configuring the device to pass the polymer through the pore from one volume to the other volume, wherein the device further comprises a sensor adjacent to the pore configured to identify objects passing through the pore; and (c) determining, with the sensor, whether the fusion molecule or particle bound to the binding motif is bound to the target molecule or particle, thereby detecting the presence of the target molecule or particle in the sample.

Claims

1. A method for detecting a target molecule or particle suspected to be present in a sample, comprising:

(a) contacting the sample with (i) a fusion molecule comprising a ligand capable of binding to the target molecule or particle and a binding domain, and (ii) a polymer scaffold comprising at least one binding motif to which the binding domain is capable of binding, under conditions that allow the target molecule or particle to bind to the ligand and the binding domain to bind to the binding motif;

(b) loading the polymer into a device comprising a pore that separates an interior space of the device into two volumes, and configuring the device to pass the polymer through the pore from one volume to the other volume, wherein the device further comprises a sensor adjacent to the pore configured to identify objects passing through the pore; and

(c) determining, with the sensor, whether the fusion molecule bound to the binding motif is bound to the target molecule or particle, thereby detecting the presence of the target molecule or particle in the sample.

2. The method of claim 1, wherein the target molecule is selected from the group consisting of a protein, a peptide, a nucleic acid, a chemical compound, an ion, and an element.

3. The method of claim 1, wherein the target particle is selected from the group consisting of a protein complex or aggregate, a protein/nucleic acid complex, a fragmented or fully assembled virus, a bacterium, a cell, and a cellular aggregate.

4. The method of claim 1, wherein step (a) is performed prior to step (b).

5. The method of claim 1, wherein step (b) is performed prior to step (a).

6. The method of claim 1, further comprising applying a condition suspected to alter the binding between the target molecule or particle and the ligand, and carrying out the determination again.

7. The method of claim 6, wherein the condition is selected from the group consisting of removing the target molecule or particle from the sample, adding an agent that competes with the target molecule or particle, or the ligand for binding, and changing the pH, salt, or temperature.

8. The method of claim 1, wherein the binding motif comprises a chemical modification for binding to the binding domain.

9. The method of claim 8, wherein the chemical modification is selected from the group consisting of acetylation, methylation, summolation, glycosylation, phosphorylation, and oxidation.

10. The method of claim 1, wherein the polymer is at least one of a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a peptide nucleic acid (PNA), a DNA/RNA hybrid, and a polypeptide.

11. The method of claim 1, wherein the binding domain is selected from the group consisting of a helix-turn-helix, a zinc finger, a leucine zipper, a winged helix, a winged helix turn helix, a helix-loop-helix, and a high mobility group box (HMG-box).

12. The method of claim 1, wherein the binding domain is selected from the group consisting of locked nucleic acids (LNAs), peptide nucleic acids (PNAs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPRs), and aptamers.

13. The method of claim 1, wherein the ligand is selected from the group consisting of an antibody, an epitope, a hormone, a neurotransmitter, a cytokine, a growth factor, a cell recognition molecule, and a receptor.

14. The method of claim 1, wherein the binding domain and the ligand are linked via an interaction selected from the group consisting of a covalent bond, a hydrogen bond, an ionic bond, a metallic bond, a van der Walls force, a hydrophobic interaction, and a planar stacking interaction, or are translated as a continuous polypeptide, to form the fusion molecule.

15. The method of claim 1, further comprising contacting the sample with a detectable label capable of binding to the target molecule or particle, or the target molecule or particle/ligand complex.

16. The method of claim 1, wherein the polymer comprises at least two units of the binding motif.

17. The method of claim 1, wherein the polymer comprises at least two different binding motifs;

the sample is in contact with at least two fusion molecules each comprising a different binding domain capable of binding to each binding motif and a different ligand capable of binding to a different target molecule or particle; and

the sensor is configured to identify whether the fusion molecule bound to each binding motif is bound to a target molecule or particle.

18. The method of claim 1, wherein the device comprises electrodes to apply a voltage differential between the two volumes.

19. The method of claim 1, wherein the device comprises an upper chamber, a middle chamber and a lower chamber,

wherein the upper chamber is in communication with the middle chamber through a first pore, and the middle chamber is in communication with the lower chamber through a second pore;

wherein the first pore and second pore are about 1 nm to about 100 nm in diameter, and are about 10 nm to about 1000 nm apart from each other; and

wherein each of the chambers comprises an electrode for connecting to a power supply.

20. The method of claim 1, wherein the device comprises an upper chamber, a middle chamber and a lower chamber,

wherein the upper chamber is in communication with the middle chamber through a first pore, and the middle chamber is in communication with the lower chamber through a second pore;

wherein the first pore and second pore are about 100 nm to about 10000 nm in diameter, and are about 10 nm to about 1000 nm apart from each other; and

wherein each of the chambers comprises an electrode for connecting to a power supply.

21. The method of claim 1, further comprising moving the polymer in a reversed direction after the binding motif passes through the pore, such as to identify, again, whether the fusion molecule bound to each binding motif is bound to a target molecule or particle.

22. A kit, package or mixture for detecting the presence of a target molecule or particle, comprising:

(a) a fusion molecule comprising a ligand capable of binding to the target molecule or particle and a binding domain;

(b) a polymer scaffold comprising at least one binding motif to which the binding domain is capable of binding; and

(c) a device comprising a pore that separates an interior space of the device into two volumes, wherein the device is configured to allow the polymer to pass through the pore from one volume to the other volume, and wherein the device further comprises a sensor adjacent to the pore configured to identify whether the binding motif is (i) bound to the fusion molecule while the ligand is bound to the target molecule or particle, (ii) bound to the fusion molecule while the ligand is not bound to the target molecule or particle, or (iii) not bound to the fusion molecule.

23. The kit, package or mixture of claim 22, further comprising a sample suspected of containing the target molecule or particle.

24. The kit, package or mixture of claim 23, wherein the sample further comprises a detectable label capable of binding to the target molecule or particle, or the target molecule or particle/ligand complex.