Single-molecule electronic multiplex nanopore immunoassays for biomarker detection

A method for detecting the presence of a compound of interest in a sample, which comprises:

• a) contacting a nanopore with a magnetic bead comprising multiple copies of a capture compound for the compound of interest attached thereto and a binding compound 2 for attachment of the magnetic bead to or near the nanopore via binding compound 1, wherein the binding compound 1 is attached to or near the nanopore, under conditions permitting the magnetic bead to attach to or near the nanopore;
• b) inserting the nanopore into an electrically resistive barrier;
• c) contacting the capture compounds on the bead with a sample containing the compound of interest under conditions permitting the compound of interest to bind to the capture compounds;
• d) contacting the compound of interest with a tagged compound under conditions permitting the tagged compound to attach to the compound of interest, wherein the tagged compound comprises at least one tag;
• e) contacting the nanopore with an electrolyte solution and applying a voltage across the electrically resistive barrier;
• and
• f) measuring the electronic signal change across the pore resulting from at least one tag of the tagged compound entering the nanopore,
• thereby detecting the presence of the compound of interest.

A method for determining the presence and/or relative quantities of a plurality of different compounds of interest in a sample, which comprises:

• a) contacting a plurality of nanopores with a plurality of different capture compounds,
• wherein each type of capture compound binds to a different type of compound of interest in the sample,
• under conditions such that each capture compound binds to or near one of the plurality of nanopores;
• b) inserting the plurality of nanopores in an electrically resistive barrier;
• c) contacting the plurality of different capture compounds with the sample comprising the plurality of different compounds of interest under conditions permitting each compound of interest to bind to a capture compound;
• d) contacting the plurality of different compounds of interest with a plurality of different tagged compounds,
• wherein each type of tagged compound binds to a different type of compound of interest,
• wherein each type of tagged compound comprises at least one tag,
• wherein each type of tagged compound comprises a different type of tag from each other type of tagged compound,
• wherein the identity of each type of tagged compound is correlated to a type of tag,
• under conditions such that each tagged compound binds to a compound of interest;
• e) contacting the plurality of nanopores with an electrolyte solution and applying a voltage across the electrically resistive barrier;
• f) measuring the electronic signal change across each of the plurality of nanopores resulting from at least one tag of each of the tagged compounds entering the nanopore;
• and
• g) optionally comparing the relative number of each type of tag so detected by a nanopore;
• thereby determining the presence and/or relative quantities of the plurality of different compounds of interest in the sample.

A method for determining the presence and/or relative quantities of a plurality of different compounds of interest in a sample, which comprises:

• a) contacting the plurality of nanopores with a plurality of different capture compounds,
• wherein each type of capture compound binds to a different type of compound of interest in the sample,
• wherein each type of capture compound comprises at least one tag,
• wherein each type of capture compound comprises a different type of tag from each other type of capture compound,
• wherein the identity of each type of capture compound is correlated to a type of tag,
• under conditions such that each capture compound binds to or near one of the plurality of nanopores;
• b) inserting the plurality of nanopores in an electrically resistive barrier;
• c) contacting the plurality of nanopores with an electrolyte solution and applying a voltage across the electrically resistive barrier;
• d) measuring the electronic signal change across each of the plurality of nanopores resulting from at least one tag of each of the capture compounds entering a nanopore, thereby determining the identity of the capture compound attached near or to each nanopore;
• e) contacting the plurality of different capture compounds with the sample comprising the plurality of different compounds of interest,
• wherein each compound of interest has the same type of tag attached thereto,
• under conditions permitting each compound of interest to bind to a capture compound;
• f) contacting the plurality of nanopores with an electrolyte solution and applying a voltage across the electrically resistive barrier;
• g) measuring the electronic signal change across each of the plurality of nanopores resulting from the tag of each of the compounds of interest entering the nanopore;
• and
• h) optionally comparing the relative number of tags detected by a nanopore;
• thereby determining the presence and/or relative quantities of the plurality of different compounds of interest in the sample.

A method for determining a molecular interaction of a biological molecule comprising:

• a) attaching the biological molecule or multiple copies of said biological molecule to or near a nanopore;
• b) inserting the nanopore in an electrically resistive barrier;
• c) contacting the biological molecule with a sample containing putative interacting compound under conditions such that the biological molecule and putative interacting compound interact, and optionally performing a cross-linking step to bind the biological molecule and putative interacting compound;
• d) contacting the putative interacting compound with a tagged compound, wherein the tagged compound comprises at least one tag, under conditions permitting the tagged compound to bind to the putative interacting compound;
• e) contacting the nanopore with an electrolyte solution and applying a voltage across the electrically resistive barrier;
• and
• f) measuring the electronic signal change across the pore resulting from at least one tag of the tagged compound entering the nanopore,
• thereby detecting the molecular interaction of a biological molecule.