US 11,685,922 B2
Aptamer method
Daniel John Turner, Oxford (GB); Daniel George Fordham, Oxford (GB); Roger Charles Gill, Oxford (GB); Clive Gavin Brown, Cambridge (GB); Stuart Reid, Oxford (GB); James Anthony Clarke, Oxford (GB); and James White, Oxford (GB)
Assigned to Oxford Nanopore Technologies PLC, Oxford (GB)
Filed by Oxford Nanopore Technologies PLC, Oxford (GB)
Filed on Jun. 30, 2020, as Appl. No. 16/916,305.
Application 16/916,305 is a continuation of application No. 14/378,929, granted, now 10,739,341, previously published as PCT/GB2013/050348, filed on Feb. 14, 2013.
Claims priority of provisional application 61/599,240, filed on Feb. 15, 2012.
Prior Publication US 2021/0018500 A1, Jan. 21, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. G01N 33/00 (2006.01); G01N 33/543 (2006.01); G01N 33/53 (2006.01); C12N 15/115 (2010.01); C07H 21/04 (2006.01)
CPC G01N 33/54386 (2013.01) [C12N 15/115 (2013.01); G01N 33/5308 (2013.01); G01N 2333/36 (2013.01); G01N 2333/49 (2013.01); G01N 2333/705 (2013.01); G01N 2333/96463 (2013.01)] 12 Claims
 
1. A multiplex assay method for determining the presence of at least one oligonucleotide aptamer bound to an analyte member of a group of two or more analytes, the method comprising:
(a) contacting a solution with a transmembrane pore, a group of two or more non-nucleic acid analytes, and a plurality of oligonucleotide aptamers, each aptamer being folded into a three-dimensional structure;
wherein each oligonucleotide aptamer in the plurality specifically binds to one of the non-nucleic acid analyte members of the group;
wherein each oligonucleotide aptamer in the plurality is conjugated to a linear polymer tail, wherein each linear polymer tail that is conjugated to an oligonucleotide aptamer in the plurality is different than each other linear polymer tail that is conjugated to an oligonucleotide aptamer in the plurality, and wherein each linear polymer tail enters the pore and affects current flow through the pore;
(b) applying an electrical potential across the pore, wherein at least two linear polymer tails each conjugated to a different oligonucleotide aptamer each enter the pore, in succession, and each affects current flow through the pore;
wherein movement of each linear polymer tail through the pore produces a distinct current flow that is different than current flow through the pore produced by the other linear polymer tails, and each oligonucleotide aptamer unfolds under the influence of the potential and then moves through the pore after its linear polymer tail;
(c) successively measuring current flowing through the pore as each linear polymer tail of step (b) enters the pore and each oligonucleotide aptamer unfolds under the influence of the potential and then moves through the pore after its linear polymer tail; and
(d) determining the presence of at least one oligonucleotide aptamer bound to a non-nucleic acid analyte member of the group of two or more non-nucleic acid analytes based on the distinct current flow through the pore caused by the linear polymer tail of step (b) and each oligonucleotide aptamer.