Nanopore biosensors for detection of proteins and nucleic acids

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.

1 - 16 . (canceled) 
     
     
         17 . A nanopore sensor comprising a modified ClyA pore in combination with a ligand wherein the ligand confers selective binding properties to an analyte. 
     
     
         18 . A nanopore sensor according to  claim 17  wherein the ligand is an aptamer, antibody, receptor and/or a peptide. 
     
     
         19 . A nanopore sensor according to  claim 18  wherein the ligand is a nucleic acid aptamer or a peptide aptamer that is selected to bind to a specific target protein analyte. 
     
     
         20 . A nanopore sensor according to  claim 19  wherein the protein analyte binds within the lumen of the modified ClyA pore. 
     
     
         21 . A nanopore sensor according to  claim 19  wherein the protein analyte is a protein with a molecular weight in the range of 15-70 kDa. 
     
     
         22 . A nanopore sensor according to  claim 20  wherein the protein analyte is a protein with a molecular weight in the range of 15-70 kDa. 
     
     
         23 . A nanopore sensor according to  claim 17  wherein the lumen of the modified ClyA pore is modified to alter the size, binding properties, and/or structure of the pore. 
     
     
         24 . A nanopore sensor according to  claim 17  wherein the nanopore is a modified ClyA pore comprising at least 12 subunits. 
     
     
         25 . A nanopore sensor according to  claim 24  wherein each subunit comprises a polypeptide represented by an amino acid sequence of SEQ ID NO: 1; SEQ ID NO: 2; or SEQ ID NO: 3. 
     
     
         26 . A method for detecting a target analyte in a sample comprising:
 a. contacting the sample with a nanopore sensor according to  claim 17 ;   b. applying one or more electric potentials across the nanopore;   c. measuring current passing through the nanopore at each of the one or more electrical potentials;   d. comparing measured currents with reference currents, wherein a change in current relative to the reference currents indicates that the target analyte is present in the sample.   
     
     
         27 . A method according to  claim 26  wherein the target analyte is a protein.