Nanopores with internal protein adaptors

Abstract

A method for detecting an analyte in a sample includes the steps of obtaining a nanopore sensor comprising a nanopore and a protein adaptor internalized in the lumen of the nanopore, adding a sample comprising an analyte to the cis side or the trans side of the nanopore, and measuring conductance across the nanopore. A change in conductance after adding the sample indicates the analyte is present in the sample and has bound to the protein adaptor. Nanopore sensors comprise a nanopore and a protein adaptor internalized in the lumen of the nanopore. The protein adaptor is a functional enzyme or ligand-binding protein.

1 .- 30 . (canceled) 
     
     
         31 . A method for detecting a ligand in a sample, comprising the steps of:
 a) obtaining a nanopore sensor comprising a biological nanopore and a protein adaptor internalized in the lumen of the nanopore, wherein the protein adaptor is a functional enzyme that is capable of binding the ligand,   b) adding the sample comprising the ligand to the cis side or the trans side of the nanopore, and   c) measuring a first conductance change across the nanopore which reflects binding of the ligand to the internalized enzyme adaptor, and measuring additional conductance changes across the nanopore which reflect enzyme kinetics such as association and dissociation of the ligand and the internalized enzyme adaptor.   
     
     
         32 . The method according to  claim 31 , wherein the nanopore has a first and a second opening whereby the first opening has a wider diameter than the second opening. 
     
     
         33 . The method according to  claim 31 , wherein the nanopore is cytolysin A (ClyA). 
     
     
         34 . The method according to  claim 31 , wherein the nanopore is the cytolysin A (ClyA) mutant Gln56Trp. 
     
     
         35 . The method according to  claim 33 , wherein ClyA comprises a plurality of subunits, each subunit comprising an amino acid sequence represented by SEQ ID NO:3. 
     
     
         36 . The method according to  claim 31 , wherein the protein adaptor is globular. 
     
     
         37 . The method according to  claim 31 , wherein the protein adaptor is a demethylase enzyme or a reductase enzyme. 
     
     
         38 . The method according to  claim 37 , wherein the demethylase is AlkB demethylase. 
     
     
         39 . The method according to  claim 37 , wherein the reductase is dihydrofolate reductase. 
     
     
         40 . The method according to  claim 31 , wherein the protein adaptor comprises a tag, wherein the tag has a net overall positive or net overall negative charge. 
     
     
         41 . The method according to  claim 31 , wherein the protein adaptor forms a complex with one or more additional molecules. 
     
     
         42 . The method according to  claim 31 , wherein the ligand is a small molecule, a protein, or a nucleic acid. 
     
     
         43 . The method according to  claim 31 , wherein the ligand is charged. 
     
     
         44 . A nanopore sensor comprising a biological nanopore and a protein adaptor internalized in the lumen of the nanopore, wherein the protein adaptor is a functional enzyme that binds a ligand while the functional enzyme is internalized, and wherein the sensor is configured to allow measurement of a first conductance change across the nanopore which reflects binding of the ligand to the internalized enzyme adaptor, and to allow measurement of additional conductance changes across the nanopore which reflect enzyme kinetics such as association and dissociation of the ligand and the internalized enzyme adaptor. 
     
     
         45 . The nanopore sensor according to  claim 44 , wherein the nanopore sensor is cytolysin A (ClyA). 
     
     
         46 . Use of a nanopore sensor according to  claim 44 , for the detection of a ligand in a sample. 
     
     
         47 . The method of  claim 38 , wherein the AlkB demethylase comprises an Asn120Asp mutation. 
     
     
         48 . Use of a nanopore sensor according to  claim 45 , for the detection of a ligand in a sample. 
     
     
         49 . The method according to  claim 37 , wherein the protein adaptor is a demethylase enzyme. 
     
     
         50 . The method according to  claim 37 , wherein the protein adaptor is a reductase enzyme.