Biosensor compositions and methods of use

Abstract

Provided are pore-subunit polypeptides covalently linked to one or more sensing moieties, and uses of these modified polypeptides to detect and/or measure analytes or physical characteristics within a given sample.

1 . A modified pore-subunit polypeptide comprising a pore-subunit polypeptide covalently linked to at least a first sensing moiety, wherein said modified pore-subunit polypeptide assembles into an oligomeric pore assembly in the presence of a plurality of pore-subunit polypeptides.  
     
     
         2 . The modified polypeptide of  claim 1 , wherein said sensing moiety is a functional group.  
     
     
         3 . The modified polypeptide of  claim 2 , wherein said functional group is an analyte-binding functional group.  
     
     
         4 . The modified polypeptide of  claim 2 , wherein said functional group is a synthetic molecule.  
     
     
         5 . The modified polypeptide of  claim 4 , wherein said functional group is a calixarene or a crown ether.  
     
     
         6 . The modified polypeptide of  claim 2 , wherein said functional group is a naturally occurring molecule.  
     
     
         7 . The modified polypeptide of  claim 6 , wherein said functional group is an enzyme inhibitor, a hapten, a nucleotide, an amino acid, a lipid, a toxin, a saccharide, a chelator or a cyclodextrin.  
     
     
         8 . The modified polypeptide of  claim 1 , wherein said sensing moiety is a polymer.  
     
     
         9 . The modified polypeptide of  claim 8 , wherein said polymer is polyethylene glycol (PEG).  
     
     
         10 . The modified polypeptide of  claim 9 , wherein said polymer is polyethylene glycol (PEG)-biotin.  
     
     
         11 . The modified polypeptide of  claim 8 , wherein said polymer is an analyte-binding polymer.  
     
     
         12 . The modified polypeptide of  claim 11 , wherein said polymer is an oligonucleotide, an oligosaccharide or a peptide.  
     
     
         13 . The modified polypeptide of  claim 1 , wherein said sensing moiety binds to a metal, metal ion, a toxin, an enzyme, a nucleotide, an oligonucleotide, an amino acid, a peptide, a saccharide, a hapten, a lipid or an antibody or antigen-binding fragment thereof.  
     
     
         14 . The modified polypeptide of  claim 1 , wherein said sensing moiety responds to a change in the type or amount of a biological or chemical constituent in the environment of said oligomeric pore assembly.  
     
     
         15 . The modified polypeptide of  claim 1 , wherein said sensing moiety responds to a change in the physical environment of said oligomeric pore assembly.  
     
     
         16 . The modified polypeptide of  claim 15 , wherein said sensing moiety responds to a change in pH, light, voltage or temperature.  
     
     
         17 . The modified polypeptide of  claim 1 , wherein said polypeptide is covalently linked to at least a first and at least a second sensing moiety.  
     
     
         18 . The modified polypeptide of  claim 17 , wherein said at least a first sensing moiety is distinct from said at least a second sensing moiety.  
     
     
         19 . The modified polypeptide of  claim 17 , wherein said at least a first sensing moiety is the same as said at least a second sensing moiety.  
     
     
         20 . The modified polypeptide of  claim 1 , wherein said polypeptide is a staphylococcal hemolysin polypeptide, a porin, a complement pore polypeptide, a hemolysin C polypeptide or a streptolysin O polypeptide.  
     
     
         21 . The modified polypeptide of  claim 20 , wherein said polypeptide is a staphylococcal alpha hemolysin polypeptide.  
     
     
         22 . The modified polypeptide of  claim 21 , wherein said polypeptide is a mutant staphylococcal alpha hemolysin polypeptide comprising at least a first heterologous amino acid.  
     
     
         23 . The modified polypeptide of  claim 22 , wherein said mutant staphylococcal alpha hemolysin polypeptide comprises a cysteine residue in place of serine at position 106 of the wild-type staphylococcal alpha hemolysin polypeptide or a cysteine residue in place of lysine at position 8 of the wild-type staphylococcal alpha hemolysin polypeptide.  
     
     
         24 - 43 . (canceled)