# US 12,214,347 B2 · Nanopore/nanowell electrode enabled exonuclease sequencing > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,214,347 B2 - Document ID: US12214347B2 - Status: Granted - Kind: B2 - Application number: 18/240,253 - Filed: 2023-08-30 - Published: 2023-12-21 - Granted: 2025-02-04 - Earliest claimed: 2017-04-19 - Assignees: Electronic Biosciences, Inc. - Applicants: Electronic Biosciences, Inc. - Inventors: Anna E. P. Schibel; Eric N. Ervin; Sean German - Web page: https://patentgrep.com/patent/US12214347B2 ## CPC - B01L 3/5027: {by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip} - C12Q 1/6869: Methods for sequencing - G01N 33/48721: {Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68)} - B01L 2300/0636: Integrated biosensor, microarrays - B01L 2300/0858: Side walls - B01L 2400/0418: electro-osmotic flow [EOF] - B01L 2400/0421: electrophoretic flow - B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Devices and methods are provided for identifying individual monomeric units in sequential order as they are released or cleaved from a polymer strand via an enzyme, which acts on the polymer, and the monomeric units translocate through a transmembrane channel. Methods are also provided for identifying molecules as they translocate through a transmembrane channel. ## Claims ### Claim 1 A method for determining a polymer sequence, comprising: a) electrophoretically and/or electroosmotically driving a polymer from a bulk solution through a transmembrane channel disposed in a membrane into a confined volume of a chamber; whereby the polymer contacts an enzyme in the confined volume of the chamber, under conditions in which the enzyme cleaves monomeric units from the polymer; b) electrophoretically and/or electroosmotically driving monomeric units in the order which they are sequentially cleaved from the polymer by the enzyme out of the confined volume of the chamber through the transmembrane channel; and c) determining the identity of each of the monomeric units based on its current signature, translocation time, and/or associated current noise level modulation as the monomeric unit translocates through the transmembrane channel, thereby determining the polymer sequence. ### Claim 2 (depends on 1) The method of claim 1, wherein the confined volume of the chamber is about 100 zeptoliters to about 1 picoliter. ### Claim 3 (depends on 2) The method of claim 2, wherein the confined volume is about 1 attoliter to about 1 femtoliter. ### Claim 4 (depends on 1) The method of claim 1, wherein the membrane comprises one or more of a planar lipid bilayer, triblock copolymer, Si, SiN, Si3N4, SiO2, glass, quartz, aluminum, kapton, paralene, polyimide, diamond, and fused silica. ### Claim 5 (depends on 1) The method of claim 1, wherein the enzyme is attached to an interior surface of the chamber at a distance from the membrane greater than about 100 nm to about 2000 nm. ### Claim 6 (depends on 5) The method of claim 5, wherein the enzyme is at a distance from the membrane of about 500 nm to about 1000 nm. ### Claim 7 (depends on 1) The method of claim 1, wherein a monomeric unit encounters and is translocated through the transmembrane channel in less than 300 milliseconds. ### Claim 8 (depends on 1) The method of claim 1, wherein: the chamber comprises a proximal opening and a distal opening and a chamber length from the proximal opening to the distal opening, and the chamber length is less than about 5 micrometers. ### Claim 9 (depends on 1) The method of claim 1, wherein electrophoretically and/or electroosmotically driving a polymer in step a comprises applying a voltage bias, and the method comprises decreasing the voltage bias after step a. ### Claim 10 (depends on 1) The method of claim 1, wherein: an enzyme capture probability time comprises a time in which the enzyme cleaves a monomeric unit from the polymer in step a; and step b is carried out after the enzyme capture probability time. ### Claim 11 (depends on 10) The method of claim 10, wherein the enzyme capture probability time is about 1 microsecond to about 10 minutes. ### Claim 12 (depends on 1) The method of claim 1, wherein electrophoretically and/or electroosmotically driving a polymer in step a comprises applying a voltage bias having a polarity, and step b comprises reversing the voltage bias polarity. ### Claim 13 (depends on 1) The method of claim 1, wherein: the enzyme cleaves a monomeric unit from the polymer at a maximum rate in the confined volume; and a monomeric unit, after being cleaved from the polymer, encounters and translocates through the transmembrane channel in a period of time less than or equal to the maximum rate. ### Claim 14 (depends on 1) The method of claim 1, wherein the polymer is single stranded RNA or single stranded DNA. ### Claim 15 (depends on 1) The method of claim 1, wherein the monomeric unit is a nucleotide monophosphate, a modified nucleotide monophosphate, a nucleoside monophosphate, a modified nucleoside monophosphate, a ribonucleotide, a deoxyribonucleotide, a modified ribonucleotide and/or a modified deoxyribonucleotide. ### Claim 16 (depends on 1) The method of claim 1, wherein the confined volume of the chamber comprises an aqueous solution having a viscosity, and the viscosity of the aqueous solution is increased or decreased to a level that increases capture efficiency and/or measurable resolution of the polymer or monomeric unit by the transmembrane channel. ### Claim 17 (depends on 1) The method of claim 1, wherein the transmembrane channel comprises a nanopore, an ion channel or and/or a transmembrane protein, and optionally the transmembrane protein comprises a biological nanopore and the biological nanopore is alpha-hemolysin (αHL), aerolysin, Mycobacterium smegmatis porin A (MspA), Escherichia coli CsgG, or outer membrane protein F (OmpF). ### Claim 18 (depends on 17) The method of claim 17, wherein the transmembrane channel comprises: (a) a mutated, engineered and/or chemically modified transmembrane channel, (b) a mutant form transmembrane channel comprising a modification of the transmembrane channel; or (c) a solid state nanopore.