阅读说明：本技术 一种核酸分子分析系统 (Nucleic acid molecule analysis system ) 是由 贾晓轻 于 2019-11-30 设计创作，主要内容包括：一种核酸分子分析系统,用于识别偶联有标签的核酸分子,包括：第一流体通道,其具有第一宽度,所述第一宽度大于所述标签直径；纳米孔,其具有第二宽度的直径,所述第二宽度小于所述标签直径,所述纳米孔下方设置第一电极；还包括第二流体通道,所述第二流体通道设置第三电极；根据通过第一电极的电流确定样本中的核酸序列；其中偶联有标签的核酸分子的标签离开所述纳米孔时的路线与所述偶联有标签的核酸分子的标签流向所述纳米孔的路线没有重叠部分,从而提高了检测效率。(A nucleic acid molecule analysis system for identifying a tag-coupled nucleic acid molecule, comprising: a first fluid channel having a first width, the first width being greater than the label diameter; a nanopore having a diameter of a second width, the second width being less than the tag diameter, a first electrode disposed below the nanopore; the device also comprises a second fluid channel, wherein the second fluid channel is provided with a third electrode; determining a nucleic acid sequence in the sample from the current through the first electrode; wherein the label of the nucleic acid molecule coupled with the label leaves the nanopore without overlapping with the label of the nucleic acid molecule coupled with the label flowing to the nanopore, thereby improving the detection efficiency.)

1. A nucleic acid molecule analysis system for identifying a tag-coupled nucleic acid molecule, comprising: a first fluid channel having a first width, the first width being greater than the label diameter; a nanopore having a diameter of a second width, the second width being less than the tag diameter, a first electrode disposed below the nanopore; the method is characterized in that: further comprising a second fluid channel having a third width; the first fluid channel is positioned on the side of the nanopore, a second electrode with unchanged polarity is arranged on one side of the first fluid channel opposite to the nanopore, and the polarity of the second electrode is opposite to that of the first electrode; the second fluid channel is located above the nanopore, the third width being greater than the tag diameter; the second fluid channel is provided with a third electrode; the first electrode is not energized when the tag-coupled nucleic acid molecule flows toward the front of the first channel of the nanopore and when the tag-coupled nucleic acid molecule exits the nanopore; the first electrode has an opposite polarity to the second electrode when the tag-coupled nucleic acid molecule flows to the back of the first fluidic channel of the nanopore; the third electrode has a polarity opposite to the second electrode when the tagged nucleic acid molecule flows toward the front of the first fluid channel of the nanopore and when the tagged nucleic acid molecule exits the nanopore; the third electrode is not energized when the tag-coupled nucleic acid molecule flows to the back of the first fluid channel of the nanopore.

2. The nucleic acid molecule analysis system of claim 1, wherein: the first fluid channel is at an angle of about 90 degrees to the nanopore.

3. The nucleic acid molecule analysis system of claim 1, wherein: the label is fluorescein imide or hexachlorofluorescein.

4. The nucleic acid molecule analysis system of claim 1, wherein: the nanopore is a biological nanopore or a solid-state nanopore.

5. The nucleic acid molecule analysis system of claim 1, wherein: the tag is coupled to the nucleic acid molecule via a linker.

6. The nucleic acid molecule analysis system of claim 1, wherein: the tag is a polypeptide.

7. The nucleic acid molecule analysis system of claim 1, wherein: the tag is coupled to the end of the nucleic acid molecule.

8. The nucleic acid molecule analysis system of claim 1, wherein: the nucleic acid molecule analysis system is used for determining a nucleic acid sequence in a sample.

9. The nucleic acid molecule analysis system of claim 1, wherein: the nucleic acid molecule is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

10. The nucleic acid molecule analysis system of claim 1, wherein: the sample is from a bodily fluid.

Technical Field

The present invention relates to a nucleic acid molecule analysis system.

Background

Nanopores are nanoscale pores embedded on biological membranes or fabricated on solid state membranes. When a voltage is applied across the nanopore, a field is formed in and around the nanopore. Ions, DNA, RNA, polypeptides, and other biological macromolecules typically carry a surface charge that passes through the nanopore under the influence of an electric field force as they diffuse into the vicinity of the nanopore. By adopting the method of detecting the current passing through the nanopore, when a substance passes through the nanopore, the change of the ionic current can be caused, so that the information of the molecules of the substance to be detected can be obtained.

In DNA sequencing, when DNA passes through a nanopore, different signals are generated through the nanopore due to the different chemical properties of each base, and the base sequence of DNA is obtained by the difference of the signals, so that rapid sequencing can be realized. However, the DNA molecule is too fast to pass through the hole, and the current change generated when a single base passes through the nanopore is difficult to capture. The main technical means in the prior art is to reduce the perforation speed of nucleic acid molecules by changing voltage or solution, such as the technical scheme proposed by the patent of CN102621214A at Beijing university, the method can improve time resolution, but the requirement of determining the current change of each base passing through a nanopore is high, and the requirement of sensitivity of the sensor is high.

CN107110817A and CN109863391A propose methods for identifying specific sequences of nucleic acid molecules, wherein a tag with a diameter larger than that of a nanopore is coupled to one end of a nucleic acid molecule, and when the tag is blocked by the nanopore, the current of an electrode near the nanopore is changed, and the specific sequence coupled to the tag is determined through the current change. However, in this method, the nanopore is blocked after it is exposed to the label, it is necessary to separate the label from the nucleic acid molecule or to apply an opposite voltage to separate the label from the nanopore in a direction opposite to the flow direction. The present invention proposes, as an improvement of CN107110817A, a nucleic acid molecule analysis system capable of improving detection efficiency.

Disclosure of Invention

The present invention provides a nucleic acid molecule analysis system for identifying a tag-coupled nucleic acid molecule, comprising: a first fluid channel having a first width, the first width being greater than the label diameter; a nanopore having a diameter of a second width, the second width being less than the tag diameter, a first electrode disposed below the nanopore; further comprising a second fluid channel having a third width; the first fluid channel is positioned on the side of the nanopore, a second electrode with unchanged polarity is arranged on one side of the first fluid channel opposite to the nanopore, and the polarity of the second electrode is opposite to that of the first electrode; the second fluid channel is located above the nanopore, the third width being greater than the tag diameter; the second fluid channel is provided with a third electrode; the first electrode is not energized when the tag-coupled nucleic acid molecule flows toward the front of the first channel of the nanopore and when the tag-coupled nucleic acid molecule exits the nanopore; the first electrode has an opposite polarity to the second electrode when the tag-coupled nucleic acid molecule flows to the back of the first fluidic channel of the nanopore; the third electrode has a polarity opposite to the second electrode when the tagged nucleic acid molecule flows toward the front of the first fluid channel of the nanopore and when the tagged nucleic acid molecule exits the nanopore; the third electrode is not energized when the tag-coupled nucleic acid molecule flows to the back of the first fluid channel of the nanopore.

Preferably, the first fluid channel is at an angle of about 90 degrees to the nanopore.

Preferably, the label is fluorescein imide or hexachlorofluorescein.

Preferably, the nanopore is a biological nanopore or a solid state nanopore.

Preferably, the tag is coupled to the nucleic acid molecule via a linker.

Preferably, the tag is a polypeptide.

Preferably, the tag is coupled to the end of the nucleic acid molecule.

Preferably, the nucleic acid molecule analysis system is used for determining a nucleic acid sequence in a sample.

Preferably, the nucleic acid molecule analysis system determines the sequence of the nucleic acid in the sample based on the current passing through the first electrode.

Preferably, the nucleic acid molecule is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

Preferably, the sample is from a bodily fluid.

Preferably, the label of the label-conjugated nucleic acid molecule leaves the nanopore without overlapping with the path of the label-conjugated nucleic acid molecule to flow to the nanopore.

The present invention also provides a nucleic acid molecule analysis system for identifying a tag-coupled nucleic acid molecule, comprising: a first fluid channel having a first width, the first width being greater than the label diameter; a nanopore having a diameter of a second width, the second width being less than the tag diameter, a first electrode disposed below the nanopore; further comprising a second fluid channel having a third width; the first fluid channel is positioned on the side of the nanopore, a second electrode with unchanged polarity is arranged on one side of the first fluid channel opposite to the nanopore, and the polarity of the second electrode is opposite to that of the first electrode; the second fluid channel is located above the nanopore, the third width being greater than the tag diameter; the second fluid channel comprises a first channel extending upwards and a second channel in the opposite direction of the first flow channel, the third electrode is arranged at the top end of the junction of the first channel and the second channel, and a fourth electrode is arranged on the side, opposite to the third electrode, of the second channel; the first electrode is not energized when the tag-coupled nucleic acid molecule exits the nanopore in the first channel; setting the polarity of the first electrode to be opposite to that of the second electrode at other moments; the third electrode is configured to have a polarity opposite to the second electrode when the tag-coupled nucleic acid molecule exits the nanopore and is located in the first channel, to have a polarity the same as the second electrode when the tag-coupled nucleic acid molecule exits the nanopore and is located in the second channel, and to not be energized after the tag-coupled nucleic acid molecule exits the second channel; the fourth electrode is configured to have a polarity opposite to the second electrode when the tag-coupled nucleic acid molecule exits the nanopore in the second channel and is not energized after the tag-coupled nucleic acid molecule exits the second channel.

Preferably, the length of the second channel is greater than 1.5 times the length of the first channel.

Preferably, the first passage length is less than 1/2 of the first fluid passage length.

Preferably, the absolute value of the voltage of the third electrode is less than the absolute value of the voltage of the second electrode when the tag-coupled nucleic acid molecule exits the nanopore in the second channel.

Preferably, the label-coupled nucleic acid molecule leaves the nanopore without an overlapping portion of its path to the nanopore.

Preferably, the nucleic acid molecule analysis system determines the sequence of the nucleic acid in the sample based on the current passing through the first electrode.

Drawings

FIG. 1 is a schematic view of a nucleic acid molecule analysis system according to a first embodiment of the present invention.

FIG. 2 is a schematic view of a nucleic acid molecule analysis system according to a second embodiment of the present invention.

Detailed Description

In order to more clearly illustrate the technical solutions of the present invention, the present invention will be briefly described below by using embodiments, and it is obvious that the following description is only one embodiment of the present invention, and for those skilled in the art, other technical solutions can be obtained according to the embodiments without inventive labor, and also fall within the disclosure of the present invention.

The nucleic acid molecule analysis system according to the first embodiment of the present invention, for identifying a sample coupled with a labeled nucleic acid molecule, is shown in fig. 1, and includes a first fluid channel 10, a nanopore 20, a second fluid channel 30, a first electrode 40, a second electrode 50, and a third electrode 60. The nucleic acid molecule is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and the label is coupled with the specific nucleic acid molecule through a connecting body, and the label can be fluorescein imide or hexachlorofluorescein or polypeptide. The nucleic acid molecule analysis system determines the nucleic acid sequence of the corresponding specific nucleic acid molecule in the sample by detection of the tag.

The nanopore 20 is a biological nanopore or a solid state nanopore, the diameter of the nucleic acid molecule is smaller than the nanopore 20, and the diameter of the nanopore 20 is smaller than the diameter of the tag so that the tag-coupled nucleic acid molecule causes a change in electrode current near the nanopore 20 when the tag is blocked by the nanopore 20 as the nucleic acid molecule passes through the nanopore 20. The first fluidic channel 10 is located lateral to the nanopore 20, and has a first width greater than the label diameter. The angle between the first fluid channel 10 and the nanopore may be set to 60 degrees to 12 degrees, and preferably may be set to about 90 degrees. A first electrode 40 is disposed below the nanopore 20, a second electrode 50 with a constant polarity is disposed on the opposite side of the first fluid channel 10 from the nanopore 20, and the polarity of the second electrode 50 is opposite to that of the first electrode 40, so that the nucleic acid molecule can be driven to move from the second electrode 50 to the first electrode 40.

A second fluid channel 30 is located above the nanopore 20 having a third width greater than the label diameter. The second fluid channel 30 is provided with a third electrode 60 on a side opposite to the nanopore 20. The first electrode 40 is not energized when the tag-coupled nucleic acid molecule flows toward the front of the first channel 10 of the nanopore 20 and when the tag-coupled nucleic acid molecule exits the nanopore 20; the first electrode 40 has a polarity opposite to that of the second electrode 50 when the tag-coupled nucleic acid molecule flows toward the rear of the first fluid channel 10 of the nanopore 20; the polarity of the third electrode 60 is opposite to the second electrode 50 when the tag-coupled nucleic acid molecule flows to the front of the first fluid channel 10 of the nanopore 20 and when the tag-coupled nucleic acid molecule exits the nanopore 20; the third electrode 60 is not energized when the tag-coupled nucleic acid molecule flows to the rear of the first fluid channel 10 of the nanopore. Thus, the label of the labeled nucleic acid molecule coupled to it leaves the nanopore 20 without overlapping the path of the label of the labeled nucleic acid molecule coupled to it flowing to the nanopore 20; while the labeled nucleic acid molecule coupled to the plugged nanopore 20 is attracted by the electric field of the third electrode 60 to leave the nanopore 20 through the second fluid channel 30, the next labeled nucleic acid molecule coupled to the nanopore 20 is attracted by the electric field of the third electrode 60 to flow in front of the first fluid channel 10, and after the labeled nucleic acid molecule coupled to the nanopore 20 is discharged through the second fluid channel 30, the next labeled nucleic acid molecule coupled to the nanopore 20 is attracted by the electric field of the first electrode 40 to flow in the rear of the first fluid channel 10 for detection; thereby improving the detection efficiency compared with the prior art.

As a further improvement, a preferred embodiment of the present invention is shown in fig. 2, and comprises a first fluid channel 10, a nanopore 20, a second fluid channel 30, a first electrode 40, a second electrode 50, a third electrode 60, and a fourth electrode 70.

The nanopore 20 is a biological nanopore or a solid state nanopore, the diameter of the nucleic acid molecule is smaller than the nanopore 20, and the diameter of the nanopore 20 is smaller than the diameter of the tag so that the tag-coupled nucleic acid molecule causes a change in electrode current near the nanopore 20 when the tag is blocked by the nanopore 20 as the nucleic acid molecule passes through the nanopore 20. The first fluidic channel 10 is located lateral to the nanopore 20, and has a first width greater than the label diameter. The angle between the first fluid channel 10 and the nanopore may be set to 60 degrees to 12 degrees, and preferably may be set to about 90 degrees. A first electrode 40 is disposed below the nanopore 20, a second electrode 50 with a constant polarity is disposed on the opposite side of the first fluid channel 10 from the nanopore 20, and the polarity of the second electrode 50 is opposite to that of the first electrode 40, so that the nucleic acid molecule can be driven to move from the second electrode 50 to the first electrode 40.

The second fluid passage 30 is wider than the label and includes a first passage 31 extending upward and a second passage 32 in the opposite direction of the first fluid passage 10, 1/2 providing a length of the first passage 31 less than the length of the first fluid passage 10, the length of the second passage 32 being greater than 1.5 times the length of the first passage 31. The third electrode 60 is arranged at the top end of the junction of the first channel 31 and the second channel 32, and the fourth electrode 70 is arranged on one side of the second channel 32 opposite to the third electrode 60; preferably, the third electrode 60 and the first channel 31 form an angle of 45 degrees, and the third electrode 60 and the second channel 32 form an angle of 45 degrees.

The first electrode 40 is not energized when the tag-coupled nucleic acid molecule exits the nanopore 20 at the first channel 31; setting its polarity opposite to the second electrode 50 at the rest of the time; the third electrode 60 is configured to have a polarity opposite to that of the second electrode 60 when the labeled nucleic acid molecule exits the nanopore 20 and is located in the first channel 31, and configured to have a polarity identical to that of the second electrode 60 when the labeled nucleic acid molecule exits the nanopore 20 and is located in the second channel 32, and to have an absolute value of a voltage smaller than that of the second electrode, and to be not energized after the labeled nucleic acid molecule exits the second channel 32; the fourth electrode 70 is configured to have a polarity opposite the second electrode 50 when the tag-coupled nucleic acid molecule exits the nanopore 20 and is located in the second channel 32, and is not energized after the tag-coupled nucleic acid molecule exits the second channel 32. In the above preferred embodiment, the data acquisition time of the nucleic acid molecule analysis system is increased by the configuration and length of the second fluid channel 30 and the corresponding fourth electrode 70, so that the first electrode 40 as the detection electrode does not acquire data only when the label is located in the first channel 31 with a shorter length.

The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and are intended to be within the scope of the invention.