Nanopore biosensors for detection of proteins and nucleic acids

• US 10,514,378 B2
• Filed: 05/21/2018
• Issued: 12/24/2019
• Est. Priority Date: 03/25/2013
• Status: Active Grant

• Current Assignee: Katholieke Universiteit Leuven
• Original Assignee: Katholieke Universiteit Leuven
• Inventors: Maglia, Giovanni, Soskine, Mikhael
• Primary Examiner(s): Li, Ruixiang
• Application Number:
  US15/984,974
• Publication Number:
  US 20180335425A1
• Time in Patent Office: 582 Days
• CPC Class Codes: C07K 14/255 Salmonella (G)
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/3278 involving nanosized elements, e.g. nanogaps or nanoparticles nanopores G01N33/48721; magnetic beads G01N27/745
• 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 33/54306 Solid-phase reaction mechanisms

Abstract

Described herein are nanopore biosensors based on a modified cytolysin protein. The nanopore biosensors accommodate macromolecules including proteins and nucleic acids, and may additionally comprise ligands with selective binding properties.

Claims

1. A nanopore sensor comprising a modified ClyA pore, wherein the modified ClyA pore comprises at least 12 subunits, each subunit comprising a polypeptide represented by an amino acid sequence with at least 80% identity to SEQ ID NO:

1 wherein the amino acid at position 103 (D) is substituted with a cysteine residue and wherein the position of the amino acid substitution is numbered with reference to an amino acid sequence as set forth in SEQ ID NO;

1, starting from the second amino acid residue.

2. The nanopore sensor according to claim 1, wherein the nanopore sensor further comprises a ligand that confers selective binding properties to an analyte.

3. The nanopore sensor according to claim 2, wherein the ligand is an aptamer.

4. The nanopore sensor according to claim 3, wherein the aptamer is a nucleic acid aptamer or a peptide aptamer that is selected to bind to a specific target protein analyte.

5. The nanopore sensor according to claim 2, wherein the ligand is an antibody.

6. The nanopore sensor according to claim 2, wherein the ligand is a receptor.

7. The nanopore sensor according to claim 2, wherein the ligand is a peptide.

8. The nanopore sensor according to claim 4, wherein the protein analyte binds within the lumen of the modified ClyA pore.

9. The nanopore sensor according to claim 4, wherein the protein analyte is a protein with a molecular weight in the range of 15-70 kDa.

10. The nanopore sensor according to claim 8, wherein the protein analyte is a protein with a molecular weight in the range of 15-70 kDa.

11. The nanopore sensor according to claim 1, wherein the lumen of the modified ClyA pore is modified to alter the size, binding properties, and/or structure of the pore.

12. The nanopore sensor according to claim 2, wherein the modified ClyA pore is attached to the ligand.

13. The nanopore sensor according to claim 12, wherein the modified ClyA pore is attached to the ligand via a disulfide linkage.

14. The nanopore sensor according to claim 12, wherein the modified ClyA pore is attached to the ligand via cross-linking.

15. The nanopore sensor according to claim 12, wherein the modified ClyA pore is attached to the ligand via chemical ligation.

16. The nanopore sensor according to claim 2, wherein the modified ClyA pore is not attached to the ligand.