# US 12,216,111 B2 · Nanopore-matched protein shuttle for molecular characterization and methodology for data analysis thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,216,111 B2 - Document ID: US12216111B2 - Status: Granted - Kind: B2 - Application number: 18/424,242 - Filed: 2024-01-26 - Published: 2024-12-05 - Granted: 2025-02-04 - Earliest claimed: 2017-07-17 - Assignees: President and Fellows of Harvard College - Applicants: President and Fellows of Harvard College - Inventors: Lene V. Hau; Jene A. Golovchenko - Web page: https://patentgrep.com/patent/US12216111B2 ## CPC - B82Y 15/00: Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors - 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)} - G01N 33/6872: {Intracellular protein regulatory factors and their receptors, e.g. including ion channels} - C12Q 1/6869: Methods for sequencing - B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES - 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) - 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 ## Abstract Systems and methods are provided for characterizing shuttle capture events in a nanopore sensor. The method first collects time-dependent current blockage signatures for at least one bias voltage. The method then identifies each signature as corresponding to a permanent or transient event. The method then generates a protein dynamics landscape (PDL) for the transient event signatures. The PDL comprises a set of histograms of nanopore current data and characterizes current through the nanopore during shuttle capture events. The method can then comprise identifying an entrance level blockage value based on the permanent event signatures. Permanent event captures can be determined by time duration which is larger than a certain threshold time value. Applying a voltage between the fluidic chambers above a threshold voltage level can be used to control that the vast majority of events are permanent. ## Claims ### Claim 1 A method to select a particular voltage to cause a particular entrance blockage level of a nanopore by a protein comprising: flowing an ionic current through an open nanopore; measuring the ionic current through the open nanopore (Io); applying one of a plurality of bias voltages across the open nanopore, causing a protein molecule to translocate through the open nanopore for each one of the plurality of the bias voltages; detecting a current blockage event by measuring the ionic current flow through the open nanopore while the protein molecule translocates through the open nanopore (It) for each one of the plurality of bias voltages; measuring duration of the current blockage event for each one of the plurality of bias voltages; classifying time-dependent current blockage signature for the plurality of bias voltages into permanent event signatures and transient event signatures; generating a protein dynamics landscape (PDL) for the transient event signatures for each of the plurality of bias voltages, the PDL comprising a collection of current blockage spectra for a set of increasing specified time values (itrans), wherein each current blockage spectrum comprises a histogram of the time spent at each current blockage level for the current blockage events that last less than the specified time value (itrans), and wherein the PDL reveals peaks corresponding to discrete current blockage levels; identifying an entrance current blockage level based on the permanent event signatures, wherein the entrance current blockage level comprises an intermediate blockage level of variable duration prior to a transition to a permanent current blockage level; selecting one of the plurality of bias voltages that corresponds to a PDL for the transient event signatures where the PDL reveals peaks at current blockage levels that are substantially equal to the entrance current blockage level. ### Claim 2 (depends on 1) The method of claim 1, wherein the classifying further comprises identifying as the transient event signatures, current blockage signatures showing a return to an initial current level. ### Claim 3 (depends on 2) The method of claim 2, wherein the classifying further comprises identifying as the permanent event signatures time-dependent current blockage signatures failing to show a return to an initial current level; optionally further comprising: for each of the plurality of bias voltages, calculating a ratio of a number of permanent protein molecule capture events to a number of transient protein molecule capture events; and based on the calculated ratios, determining a threshold bias voltage to achieve a preferred ratio. ### Claim 4 (depends on 1) The method of claim 1, wherein the current blockage spectra further comprises an average of nanopore current blockage data, sampled over a fixed time interval to yield histogram data points; optionally wherein the fixed time interval is at least as large as an interval for sampling the nanopore current blockage data. ### Claim 5 (depends on 1) The method of claim 1, wherein the identifying comprises: generating an additional PDL for the permanent event signatures for at least one of the plurality of bias voltages; and selecting the entrance current blockage level to correspond to a current blockage level lower than the permanent blockage level; optionally wherein the method further comprises: fitting each current blockage spectrum of the collection of current blockage spectra within a PDL with a sum of Gaussian distribution; and determining the entrance current blockage level from a peak in the collection of blockage spectra within the PDL for permanent events. ### Claim 6 (depends on 1) The method of claim 1, wherein the specified time value (itrans) is at least one of: 2 milliseconds, 5 milliseconds, 10 milliseconds, 30 milliseconds, 100 milliseconds, 500 milliseconds, and 1000 milliseconds. ### Claim 7 A method for characterizing a protein using protein capture in a nanopore sensor, comprising: a) flowing an ionic current through an open nanopore; b) measuring the ionic current through the open nanopore (Jo); c) applying a fixed bias voltage across the open nanopore, causing a protein molecule to translocate through the open nanopore; d) detecting a current blockage event by measuring the ionic current flow through the open nanopore while the protein molecule translocates through the open nanopore (It); e) measuring duration of the current blockage event; f) generating a protein dynamics landscape (PDL), the PDL comprising current blockage spectra for a set of increasing specified time values (itrans), wherein each current blockage spectrum comprises a histogram of the time spent at each current blockage level for the current blockage events that last less than the specified time value; and g) from the PDL, identifying peaks corresponding to one or more discrete current blockage levels. ### Claim 8 (depends on 7) The method of claim 7, wherein the method further comprises determining whether the duration of the protein capture event falls in a range between a pre-defined minimum duration and a pre-defined maximum duration. ### Claim 9 (depends on 7) The method of claim 7, wherein the method further comprises identifying a particular blockage event as permanent if the duration of the particular blockage event is larger than a threshold time. ### Claim 10 (depends on 9) The method of claim 9, wherein the threshold time is any period of time larger than 1000 milliseconds. ### Claim 11 (depends on 8) The method of claim 8, wherein the pre-defined minimum duration is 0 milliseconds and the pre-defined maximum duration is 2 milliseconds, optionally wherein the plurality of time-dependent current blockage spectra provides a blockage peak at 40% current blockage. ### Claim 12 (depends on 8) The method of claim 8, wherein the pre-defined minimum duration is 0 milliseconds and the pre-defined maximum duration is 10 milliseconds, optionally wherein the plurality of time-dependent current blockage spectra provides blockage peaks at 40%, 45%, and 52% current blockage. ### Claim 13 (depends on 8) The method of claim 8, wherein the pre-defined minimum duration is 0 milliseconds and the pre-defined maximum duration is 100 milliseconds, optionally wherein the plurality of time-dependent current blockage spectra provides blockage peaks at 40%, 45%, 52%, and 57% current blockage. ### Claim 14 (depends on 8) The method of claim 8, wherein the pre-defined minimum duration is 0 milliseconds and the pre-defined maximum duration is 1000 milliseconds, optionally wherein the plurality of time-dependent current blockage spectra provides a blockage peak at 80% current blockage. ### Claim 15 (depends on 8) The method of claim 8, further comprising identifying an expected protein molecule orientation based on at least one of a comparison of the PDLs for transient and permanent events and a presence or absence of blockage peaks for individual spectra with the PDLs for transient and permanent events. ### Claim 16 A method for characterizing protein molecule capture in a nanopore sensor, comprising: applying a plurality of bias voltages, the applied bias voltage polarity generates a current, which causes the protein molecule to be electrically driven into the nanopore; detecting a current blockage event by measuring the ionic current flow through the nanopore while the protein molecule translocates through the open nanopore for each one of the plurality of bias voltages; measuring duration of the current blockage event for each one of the plurality of bias voltages: classifying a plurality of time-dependent current blockage signatures for the plurality of bias voltages into permanent event signatures and transient event signatures, wherein any one of the plurality of time-dependent current blockage signatures showing a return to an initial current level, i.e., the protein molecule is trapped by and subsequently escape from the nanopore is identified as the transient event signatures, and any one of the plurality of time-dependent current blockage signatures failing to show a return to an initial current level, i.e., the protein molecule is permanently captured to the deepest level in the nanopore from which escape eventually becomes impossible is identified as the permanent event signatures; for each of the plurality of bias voltages, generating a protein dynamics landscape for the transient event signatures, wherein the protein dynamics landscape comprises a collection of blockage spectra, wherein each of the collection of blockage spectra comprises a histogram of blockage values for events of a selected maximum duration; identifying a permanent level blockage value based on the permanent event signatures; selecting one of the plurality of bias voltages corresponding to a protein dynamics landscape for the transient event signatures where the collection of blockage spectra reveals peaks at blockage values that are equal to the permanent level blockage value. ### Claim 17 (depends on 16) The method of claim 16, wherein the time-dependent current blockage signature comprises a measurement of nanopore current as a function of time during a protein molecule capture event. ### Claim 18 (depends on 16) The method of claim 16, further comprising: for each of the plurality of bias voltages, calculating a ratio of a number of permanent protein molecule capture events to a number of transient protein molecule capture events; and based on the calculated ratios, determining a threshold bias voltage to achieve a preferred ratio. ### Claim 19 (depends on 16) The method of claim 16, wherein the blockage spectra further comprises an average of nanopore current blockage data, sampled over a fixed time interval to yield histogram data points. ### Claim 20 (depends on 19) The method of claim 19, wherein the fixed time interval is at least as large as an interval for sampling the nanopore current blockage data. ### Claim 21 (depends on 19) The method of claim 19, wherein the nanopore current blockage data comprises a ratio of a change in the current from an input current to the input current. ### Claim 22 (depends on 16) The method of claim 16, wherein the identifying comprises: generating an additional protein dynamics landscape for the permanent event signatures for at least one of the plurality of bias voltages; and selecting an entrance level blockage value to correspond to a blockage level lower than the permanent blockage level. ### Claim 23 (depends on 22) The method of claim 22, wherein the method further comprises: fitting each spectrum of the collection of blockage spectra within a protein dynamics landscape with a sum of Gaussian distribution; determining the entrance level blockage value from a peak in the collection of blockage spectra within the protein dynamics landscape for permanent events. ### Claim 24 (depends on 16) The method of claim 16, wherein the selected maximum duration of the particular blockage spectra is at least one of: 2 milliseconds, 5 milliseconds, 10 milliseconds, 30 milliseconds, 100 milliseconds, 500 milliseconds, and 1000 milliseconds. ### Claim 25 (depends on 16) The method of claim 16, wherein the protein molecule comprises a protein shuttle comprising an electrically charged protein molecule linked to a target molecule.