Nanopore-matched protein shuttle for molecular characterization and methodology for data analysis thereof

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 (I0);
• 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 (τtrans), 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 the specified time value (τtrans), 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.

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 (I0);
• 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 (τtrans), 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.

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.