Method for determining biopolymer using nanopore, and system and kit therefor

Patent metadata

Field	Value
Publication number	US-10365287-B2
Application number	US-201013148479-A
Country	US
Kind code	B2
Filing date	Mar 1, 2010
Priority date	Mar 30, 2009
Publication date	Jul 30, 2019
Grant date	Jul 30, 2019

Abstract

Although analysis can be very quickly conducted at a low cost by a method for measuring a biopolymer using a nanopore, the accuracy of distinguishing the individual monopolymers constituting the biopolymer is low. To both ends of a biopolymer through a nanopore, molecules which are larger than the nanopore are attached and then the biopolymer is reciprocated by an external force to thereby perform repeated measurements.

First claim

The invention claimed is:

1. A system for determining an alignment of monomers constituting a biopolymer, wherein the system comprises:
a first solution vessel and a second solution vessel partitioned by a membrane;
a pair of electrodes configured to move the biopolymer between the first solution vessel and the second solution vessel through the nanopore, wherein the pair of electrodes in the first solution vessel and the second solution vessel is connected to a voltage source configured to bring about a voltage gradient by the electrodes between the first solution vessel and the second solution vessel;
a first introduction port configured to introduce the biopolymer, which has a first stopper molecule bound to one end of the biopolymer that is an object of measurement, into the first solution vessel, wherein the first stopper molecule is larger than a diameter of the nanopore;
a second introduction port configured to introduce a second stopper molecule, which is bound to the other end of the biopolymer having passed through the nanopore, into the second solution vessel, wherein the second stopper molecule is larger than a diameter of the nanopore;
an ammeter operatively connected to the pair of electrodes, wherein the ammeter detects a signal generated along with the movement of the biopolymer made by the pair of electrodes; and
a data processor programmed to:
apply a voltage gradient between the pair of electrodes to cause the biopolymer to migrate from the second solution vessel to the first solution vessel for a first interval of time;
automatically switch polarity of the pair of electrodes when the ammeter measures a decrease in current passing though the nanopore that is caused by approach of the first or second stopper molecule to the nanopore;
apply a voltage gradient between the pair of electrodes to cause the biopolymer to migrate from the first solution vessel to the second solution vessel for a second interval of time;
measure a temporal change in the signal detected by the ammeter;
calculate the signal as data dependent on species of monomers constituting the biopolymer; and
determine the alignment of the monomers constituting the biopolymer.

2. The system of claim 1 , wherein the biopolymer has a specific monomer thereof labeled with a fluorescent substance and wherein the system further comprises:
a light source configured to irradiate light to the biopolymer which passes through the nanopore; and
a charge-coupled device (CCD), wherein the charge-coupled device detects light emitted due to the irradiation of light.

3. The system of claim 1 , further comprising:
a set of known-sequence probes, wherein the biopolymer is a single-stranded nucleic acid; and
a third introduction port, wherein the third induction port introduces the known-sequence probe into at least one of the first and second solution vessels; and
wherein the data processor is further programmed to measure a temporal change in a signal of presence or absence of the known-sequence probe, which is bound to the single-stranded nucleic acid, along with the movement of the single-stranded nucleic acid.

4. The system of claim 1 , wherein at least one of the first and second stopper molecules is streptavidin.

5. The system of claim 4 , wherein at least one of the first and second stopper molecules is a bead to be bound to the biopolymer by a DIG-anti-DIG antibody bond.

6. The system of claim 1 , wherein the data processor is further programmed to calculate, for each interval of time, a position of the monomers on the biopolymer, and determine the alignment of the monomers constituting the biopolymer based on the calculated positions of the monomers.

7. The system of claim 1 , wherein the data processor is further programmed to repeat switching the polarity of the pair of electrodes and apply a voltage gradient between the pair of electrodes to cause the biopolymer to migrate between the first solution vessel and the second solution vessel.

8. The system of claim 1 , wherein the first interval of time is different from the second interval of time.

9. A system, comprising:
a first solution vessel having an introduction port, wherein the introduction port introduces a biopolymer and a first stopper molecule, wherein the first stopper molecule is bound to one end of the biopolymer;
a second solution vessel having an introduction port configured to introduce a second stopper molecule, wherein the second stopper molecule is bound to another end of the biopolymer;
a membrane partitioning the first and second solution vessels, the membrane having a nanopore configured to be large enough to allow portions of biopolymer between the ends to pass therethrough and smaller than the first and second stopper molecules to hinder the first and second stopper molecules from passing therethrough, the membrane formed with an insulator membrane comprising at least one of Si 3 N 4 , a plastic material, or a metallic material, and the insulator membrane comprising the nanopore of between approximately 0.5 nm and approximately 50 nm in diameter formed therein;
a pair of electrodes operatively connected to a voltage source, the electrodes having ends provided in the first and second solution vessels, and wherein the pair of electrodes bring about a voltage gradient between the first and second solution vessels to move portions of biopolymer between the ends between the first solution vessel and second solution vessel through the nanopore in the membrane;
an ammeter, wherein the ammeter detects a signal generated along with the movement of the biopolymer made by the pair of electrodes, including detecting a current passing through the nanopore;
a data processor programmed to:
apply a voltage gradient between the pair of electrodes to cause the biopolymer to migrate from the second solution vessel to the first solution vessel for a first interval of time;
automatically switch polarity of the pair of electrodes when the ammeter measures a decrease in current passing though the nanopore that is caused by approach of the first or second stopper molecule to the nanopore;
apply a voltage gradient between the pair of electrodes to cause the biopolymer to migrate from the first solution vessel to the second solution vessel for a second interval of time;
measure a temporal change in the signal detected by the ammeter;
calculate the signal as data dependent on species of monomers constituting the biopolymer; and
determine the alignment of the monomers constituting the biopolymer.

10. The system of claim 9 , wherein at least one of the first and second stopper molecules is streptavidin.

11. The system of claim 9 , wherein at least one of the first and second stopper molecules is a bead to be bound to the biopolymer by a DIG-anti-DIG antibody bond.

12. The system of claim 9 , wherein the biopolymer has a specific monomer thereof labeled with a fluorescent substance, and wherein the system further comprises:
a light source, wherein the light source irradiates light to the biopolymer which passes through the nanopore, and wherein the detector detects light emitted due to the irradiation of light.

13. A system, comprising:
a first solution vessel having an introduction port configured to introduce a biopolymer and a first stopper molecule, wherein the first stopper molecule is bound to one end of the biopolymer;
a second solution vessel having an introduction port configured to introduce a second stopper molecule, wherein the second stopper molecule is bound to another end of the biopolymer;
a membrane partitioning the first and second solution vessels, the membrane having a nanopore confi

Assignees

• Sakai Tomoyuki
• Fujita Takeshi
• Hitachi High Tech Corp

Inventors

• Sakai Tomoyuki
• Fujita Takeshi

Classifications

• G01N33/6803 Primary

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• G01N33/48721

  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) · CPC title