Device, tunneling current measuring apparatus, nucleic acid sequence reading apparatus, tunneling current measuring method, and nucleic acid sequence reading method

Abstract

To provide a device allowing a sample to easily enter a sample measurement flow path in which measurement electrodes are disposed.SOLUTION: A device used for measuring a tunnel current includes: a base material; a flow path formed in the base material; and a pair of measurement electrodes for measuring a tunnel current when a sample passes. The flow path includes: a sample supply flow path; a sample measurement flow path in which measurement electrodes are disposed; a first taper flow path that is disposed between the sample supply flow path and the sample measurement flow path, and allows flow path width to become narrower toward the sample measurement flow path from the sample supply flow path; and a sample collection flow path for collecting a sample that passes the sample measurement flow path. The width of a connection part between the first taper flow path and the sample measurement flow path ranges from 20 nm to 200 nm.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] The disclosure in this application relates to a device, a tunneling current measurement apparatus, a nucleic acid sequence reading apparatus, a tunneling current measurement method, and a nucleic acid sequence reading method. [Background technology]

[0002] Memories using DNA, a biopolymer, have attracted attention due to their high storage density and record retention stability. Commercialization of DNA memory requires technology to read DNA sequences at high speed. One known method for reading DNA sequences is to use optical probes to detect the extension reaction during the PCR amplification process. This type of device, known as a sequencer, has already been commercialized. However, this method faces the challenge of requiring an amplification process such as PCR, making it impossible to achieve reading speeds that exceed the associated extension time (approximately one second per base). Furthermore, because PCR is only effective for DNA, it cannot be applied to biomolecules other than DNA or artificial bases, which limits the storage density that can be achieved with combinations of only four bases.

[0003] As a nucleic acid reading device (method) other than PCR, a method is known in which a nanopore (a minute through-hole) is formed in a thin film and the tunneling current generated when a nucleic acid passes through the nanopore is measured (see Patent Document 1).

[0004] Furthermore, when reading a nucleic acid, it is desirable to elongate the nucleic acid. As a technique for elongating a nucleic acid, for example, a technique is known in which a channel with nanowires formed in a substrate is provided and the nucleic acid is elongated by passing through the nanowires (see Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2017-509899 [Patent Document 2] Japanese Patent Application Laid-Open No. 2016-103979 Summary of the Invention [Problem to be solved by the invention]

[0006] It is desirable for a DNA memory reading device (reading method) to improve the nucleic acid reading speed and be able to read nucleic acid sequences even with a small amount of sample. The reading device (reading method) described in Patent Document 1 forms a chamber with a thin film on which a nanopore is formed as a boundary, and measures the tunneling current when nucleic acid introduced into the chamber passes through the nanopore. However, the opening of the nanopore is formed so as to be approximately perpendicular to the thin film, and the area of ​​the opening relative to the thin film is very small. Therefore, the sample solution containing nucleic acid comes into contact with the entire thin film, which creates the problem of making it difficult for the nucleic acid in the sample solution to enter the opening.

[0007] An object of the disclosure in this application is to provide a device for solving the above problems, a tunneling current measurement apparatus and method using the device, and a nucleic acid sequence reading apparatus and method. [Means for solving the problem]

[0008] The disclosure of the present application relates to the following devices, tunneling current measurement apparatus, nucleic acid sequence reading apparatus, tunneling current measurement method, and nucleic acid sequence reading method.

[0009] (1) A device used to measure tunneling current, the device comprising: A substrate; a channel formed in a substrate; a pair of measurement electrodes for measuring the tunneling current when the sample passes through; Including, The flow path is a sample input channel; a sample measurement flow channel in which a measurement electrode is disposed; a first tapered channel disposed between the sample introduction channel and the sample measurement channel, the channel width of which narrows from the sample introduction channel toward the sample measurement channel; a sample collection flow path for collecting the sample that has passed through the sample measurement flow path; Including, The width of the connection between the first tapered channel and the sample measurement channel is 20 nm to 200 nm. device. (2) When the width of the connection portion between the first tapered channel and the sample measurement channel is defined as W1 and the width of the connection portion between the first tapered channel and the sample introduction channel is defined as W2, W2 / W1 is 10 to 20; When the length of the connecting portion between the first tapered channel and the sample measurement channel and the connecting portion between the first tapered channel and the sample introduction channel is defined as L1, L1 / W2 is 0.5 to 5. The device described in (1) above. (3) The length of the sample measurement channel is 20 nm to 1000 nm. The device according to (1) or (2) above. (4) A second tapered channel is disposed between the sample measurement channel and the sample recovery channel, and the channel width increases from the sample measurement channel toward the sample recovery channel. The device according to any one of (1) to (3) above. (5) The width of the connection portion between the sample measurement channel and the second tapered channel is defined as W1a; The width of the connection portion between the second tapered channel and the sample collection channel is defined as W2a, When the length of the connection part between the second tapered channel and the sample measurement channel and the connection part between the second tapered channel and the sample recovery channel is defined as L1a, The relationships W1=W1a, W2=W2a, and L1=L1a are satisfied. The device according to any one of (1) to (4) above. (6) A device comprising the device according to any one of (1) to (5) above, a power supply for electrophoresis, and a measurement unit, The electrophoresis power supply applies a voltage of 10 mV to 5 V to the electrophoresis electrodes. Tunnel current measurement device. (7) A tunneling current measuring device according to (6) above, including an analyzing unit, the sample is a nucleic acid, The analysis unit identifies the nucleic acid sequence from the tunneling current measurement results obtained by the tunneling current measurement device. Nucleic acid sequence reading device. (8) A tunneling current measurement method using a device, comprising: The device is A substrate; a channel formed in a substrate; a pair of measurement electrodes for measuring the tunneling current when the sample passes through; Including, The flow path is a sample input channel; a sample measurement flow channel in which a measurement electrode is disposed; a first tapered channel disposed between the sample introduction channel and the sample measurement channel, the channel width of which narrows from the sample introduction channel toward the sample measurement channel; a sample collection flow path for collecting the sample that has passed through the sample measurement flow path; Including, The width of the connection portion between the first tapered channel and the sample measurement channel is 20 nm to 200 nm, The tunnel current measurement method is as follows: a sample electrophoresis step of electrophoresing the sample in the sample input channel toward the sample recovery channel by applying a voltage to the sample input channel and the sample recovery channel; a measurement step of measuring a tunnel current when a sample passes through a gap between a pair of measurement electrodes arranged in a sample measurement flow path; A tunneling current measurement method comprising: (9) During the sample electrophoresis process, a voltage of 10 mV to 5 V is applied. The tunnel current measuring method according to (8) above. (10) When the width of the connection portion between the first tapered channel and the sample measurement channel is defined as W1 and the width of the connection portion between the first tapered channel and the sample introduction channel is defined as W2, W2 / W1 is 10 to 20; When the length of the connecting portion between the first tapered channel and the sample measurement channel and the connecting portion between the first tapered channel and the sample introduction channel is defined as L1, L1 / W2 is 0.5 to 5. The tunnel current measuring method according to (8) or (9) above. (11) The sample is a nucleic acid; a nucleic acid sequence reading step for identifying a nucleic acid sequence from the tunneling current measurement result obtained by the measurement step of the tunneling current measurement method according to any one of (8) to (10) above; A method for reading a nucleic acid sequence, comprising: [Effects of the Invention]

[0010] The devices disclosed in this application facilitate sample entry into the sample measurement channel where the measurement electrodes are located. [Brief explanation of the drawings]

[0011] [Figure 1A] FIG. 1A is a top view of device 1. [Figure 1B] FIG. 1B is a cross-sectional view taken along the line XX in FIG. 1A. [Figure 1C] FIG. 1C is a cross-sectional view taken along the line YY in FIG. 1A. [Figure 2] FIG. 2 is a schematic diagram showing an example of a procedure for producing the device 1. [Figure 3] FIG. 3 is a schematic diagram showing an outline of an embodiment of a tunneling current measurement device 100. As shown in FIG. [Figure 4] FIG. 4 is a schematic diagram showing an outline of an embodiment of a nucleic acid sequence reading apparatus 100a. [Figure 5] FIG. 5 is a flowchart of the tunneling current measurement method and the nucleic acid sequence reading method. [Figure 6] Fig. 6A is an SEM photograph of the device 1 produced in Example 1, showing the measurement electrode 4 and the vicinity of the sample measurement flow path 32 in which the measurement electrode 4 is arranged. Fig. 6B is a photograph of the device 1 with the cover member 5 adhered thereto and the electrophoresis electrode inserted therein. [Figure 7]FIG. 7 is a chart showing the results of measuring the tunneling current of the nucleic acid measured in Example 4. [Figure 8] FIG. 8 is a chart showing the results of measuring the tunnel current of nucleic acid measured in Example 5 and Comparative Example 1. [Figure 9] FIG. 9 is a graph showing the measurement results of the migration rate of nucleic acid in Example 6. [Figure 10] FIG. 10 is a graph showing the measurement results of the sample migration speed measured in Example 7. [Figure 11] FIG. 11 is a graph analyzing the measurement results of FIG. DETAILED DESCRIPTION OF THE INVENTION

[0012] The device, the tunneling current measuring apparatus, the nucleic acid sequence reading apparatus, the tunneling current measuring method, and the nucleic acid sequence reading method will be described in detail below with reference to the drawings.

[0013] In this specification, components having the same functions are denoted by the same or similar reference numerals, and repeated descriptions of components denoted by the same or similar reference numerals may be omitted.

[0014] In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits. Numerical values, numerical ranges, and qualitative expressions (e.g., expressions such as "same" and "approximately") should be interpreted as indicating numerical values, numerical ranges, and properties that include errors generally accepted in the technical field.

[0015] Furthermore, for ease of understanding, the position, size, range, etc. of each component shown in the drawings may not represent the actual position, size, range, etc. Therefore, the disclosure in this application is not necessarily limited to the position, size, range, etc. disclosed in the drawings.

[0016] (First embodiment of the device) A device 1 according to a first embodiment will be described with reference to Figures 1A to 1C. Figure 1A is a top view of the device 1, Figure 1B is a cross-sectional view taken along the line XX in Figure 1A, and Figure 1C is a cross-sectional view taken along the line YY in Figure 1A. Figure 2 is a schematic diagram showing an example of a procedure for producing the device 1.

[0017] The device 1 includes a substrate 2, a flow channel 3 formed in the substrate 2, and a pair of measurement electrodes 4a and 4b (hereinafter, the pair of measurement electrodes 4a and 4b may be referred to as "measurement electrodes 4") for measuring the tunneling current when a sample passes through. The flow channel 3 includes a sample introduction flow channel 31, a sample measurement flow channel 32 in which the measurement electrode 4 is arranged, a first tapered flow channel 33 arranged between the sample introduction flow channel 31 and the sample measurement flow channel 32 and whose flow channel width narrows from the sample introduction flow channel 31 toward the sample measurement flow channel 32, and a sample recovery flow channel 34 for recovering the sample that has passed through the sample measurement flow channel 32. The width W1 of the connection between the first tapered flow channel 33 and the sample measurement flow channel 32 is 20 nm to 200 nm.

[0018] 1A shows the second tapered channel 35, but the second tapered channel 35 is an optional component in the device 1 according to the first embodiment. If the sample flowing out of the sample measurement channel 32 can be collected, the sample collection channel 34 may be directly connected to the sample measurement channel 32.

[0019] The device 1 can be manufactured using, for example, a nanochannel-integrated mechanically controllable break junction (MCBJ) method. An example of a manufacturing procedure for the device 1 is shown with reference to FIG. 2. The mechanical break junction (MCBJ) method for fabricating a pair of measuring electrodes 4 is described, for example, in JP-A-2019-525766, the above-mentioned Patent Document 1, M. Tsutsui, K., Shoji, M. Taniguchi, T. Kawai, Nano Lett., 345 (2008), and M. Tsutsui, M. Taniguchi, T. Kawai, Appl. Phys. Lett. 93, 163115 (2008), etc.

[0020] (1) An insulating layer 2b is formed on a substrate 2a made of silicon or the like, using an insulating material such as polyimide. (2) A metal layer for forming the measuring electrode 4 is deposited on the insulating layer 2b by electron beam lithography (EB lithography). (3) A deposition layer 2c of SiO2 or the like is formed by chemical vapor deposition. A resist layer 2d is laminated on the deposition layer 2c by spin coating. (4) Using electron beam lithography, a pattern of the flow channel 3 including the sample measurement flow channel 32 is formed so as to be superimposed on the metal layer for forming the measurement electrode 4 . (5) The flow path 3 is formed by dry etching. Then, a gap (nano-gap G) is formed in the metal layer using MCBJ to form the measurement electrode 4. In the example shown in FIG. 2, the sample measurement flow path 32 is etched down to below the measurement electrode 4, but the flow path below the measurement electrode 4 does not have to be present. In addition, although there is one measurement electrode 4 in the example shown in FIG. 2, two or more measurement electrodes 4 may be formed. (6) A cover member 5 having holes formed therein as necessary for introducing a sample liquid, inserting an electrophoresis electrode, etc. is attached. The cover member 5 only needs to be attached when measuring the ion current.

[0021] There are no particular limitations on the material of the substrate 2a as long as it is a material commonly used in the field of semiconductor manufacturing technology. Examples of the material of the substrate 2a include Si, SiO x , SiN x , Ge, Se, Te, GaAs, GaP, GaN, InSb, InP, etc.

[0022] The insulating layer 2b is also not particularly limited as long as it is a material commonly used in the field of semiconductor manufacturing technology. Examples of materials for the insulating layer 2b include insulating polymers such as polyimide, polypropylene, polyvinyl chloride, polystyrene, high density polyethylene (HDPE), polyacetal (POM), and polyepoxy; insulating oxide semiconductor metals such as SiO2 and aluminum oxide; and the like.

[0023] Materials for forming the deposition layer 2c include insulating polymers such as polyimide, polypropylene, polyvinyl chloride, polystyrene, high density polyethylene (HDPE), polyacetal (POM), and polyepoxy; insulating oxide semiconductor metals such as SiO2 and aluminum oxide; and the like.

[0024] There are no particular limitations on the material for forming the measurement electrode 4 as long as it can measure tunneling current, and examples include gold, platinum, silver, palladium, tungsten, and alloys of the above metals.

[0025] There are no particular limitations on the photoresist used in electron beam lithography and the reagents used for development and etching, etc., as long as they are materials commonly used in the field of microfabrication technology. There are also no particular limitations on the equipment used in spin coaters and etching, etc., as long as they are materials commonly used in the field of microfabrication technology.

[0026] There are no particular limitations on the material of the cover member 5 as long as it can be attached to the base material 2 on which the flow path 3 is formed. Examples of materials for the cover member 5 include polymethyldisiloxane (PDMS). The cover member 5 and the base material 2 may be attached to each other by, for example, ozone plasma treatment or the like.

[0027] In this specification, the term "substrate" refers to a material portion that serves as the base for forming the flow path 3. In the example shown in FIG. 2, the substrate 2 includes a substrate 2a, an insulating layer 2b, a deposition layer 2c, and a resist layer 2d. Note that FIG. 2 is merely an example of a procedure for fabricating a device 1 in which a measurement electrode 4 is disposed in a sample measurement flow path 32. Other processes may be added or removed from the device 1 as long as the effects disclosed in this application are achieved. For example, the resist layer 2d may be removed after the flow path 3 is formed by etching. In this case, the resist layer 2d is not included in the substrate 2. The device 1 may also be fabricated by electron beam engraving, nanoprinting, or the like.

[0028] For ease of understanding, in the example shown in FIGS. 1A to 1C, detailed descriptions of the substrate 2a, insulating layer 2b, deposition layer 2c, and resist layer 2d are omitted, and they are referred to as a base material 2.

[0029] By applying a voltage to the sample input channel 31 and the sample recovery channel 34, an electrophoretic force is applied to the sample, increasing the sample's migration speed. As a result, the sample measurement speed is improved compared to when no electrophoretic force is applied. On the other hand, when applying a voltage to the channel 3 to apply an electrophoretic force to the sample, a larger voltage is required as the cross-sectional area of ​​the channel 3 increases.

[0030] Incidentally, nucleic acid reading using tunnel current is performed by identifying differences in measured picoampere-level current values. Based on the results of various experiments, the present inventors have newly discovered that when a voltage of a level sufficient to impart an electrophoretic force to nucleic acids introduced into a micrometer-order flow channel as described in Patent Document 2 is applied, the measurement electrode 4 detects noise caused by the electrophoretic voltage, making it impossible to identify the nucleic acids. The disclosure of the present application is based on this finding.

[0031] The device 1 disclosed in the present application can apply an electrophoretic force to a sample at a low voltage, allowing for measurement of tunneling current with little noise due to the electrophoretic voltage. Therefore, as described above, it can be suitably used for identifying nucleic acids, but the sample is not limited to nucleic acids. Any sample that has a surface charge and migrates by electrophoresis can be used for measurement of tunneling current with little noise due to the electrophoretic voltage. Examples of samples include peptides, lipids, sugar chains, synthetic polymers, etc. In this specification, the term "sample liquid" refers to a liquid in which the sample is dissolved or dispersed in a solvent for electrophoresis.

[0032] A sample introduction channel 31 of a predetermined size is required for introducing the sample liquid into the device 1. Therefore, the device 1 has a structure in which the width of the sample measurement channel 32 in which the measurement electrode 4 is arranged is narrowed (made small), and the sample introduction channel 31 and the sample measurement channel 32 are connected by a first tapered channel 33.

[0033] As described above, a narrow width of the sample measurement channel 32 is preferable to reduce noise caused by the voltage used for electrophoresis. When the width W1 of the connection between the first tapered channel 33 and the sample measurement channel 32 is defined, W1 may be 200 nm or less, 180 nm or less, 160 nm or less, 140 nm or less, 120 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, or 50 nm or less. Meanwhile, there is no particular lower limit for W1 as long as it is within a manufacturable range. While not limited, it may be, for example, 20 nm or more, 25 nm or more, or 30 nm or more.

[0034] The width of the sample measurement channel 32 may be the same at all points, or may vary as long as it does not affect the analysis of the measurement results. In the example shown in Figure 1A, when the end of the sample measurement channel 32 opposite to W1 is defined as W1a, W1a may be the same as W1, or may be larger or smaller than W1.

[0035] The gap between the pair of measurement electrodes 4a and 4b (gap G, see FIG. 1B) is not particularly limited as long as it is within a range that allows measurement of the tunneling current when a sample passes through. It is not particularly limited, but may be, for example, 0.1 nm or more, 0.3 nm or more, 0.5 nm or more, 0.7 nm or more, or 0.9 nm or more. Meanwhile, the upper limit of gap G is not particularly limited, but may be, for example, 50 nm or less, 30 nm or less, 10 nm or less, 8 nm or less, 6 nm or less, 5 nm or less, 4 nm or less, 3 nm or less, 2 nm or less, or 1 nm or less.

[0036] The length of the measurement electrode 4 (the length of the gap G in the same direction as L2 in FIG. 1A) is not particularly limited as long as it is within a range in which the tunneling current when the sample passes through can be measured. For example, the length may be, but is not limited to, 1000 nm or less, 800 nm or less, 600 nm or less, 400 nm or less, 200 nm or less, 100 nm or less, 80 nm or less, or 60 nm or less.

[0037] In order to facilitate cutting of the MCBJ, it is preferable that the deposition amount of the measuring electrode 4 (in the direction perpendicular to the longitudinal direction of the measuring electrode 4, or in the H direction in FIG. 1B; hereinafter, this may be referred to as "thickness") be small. Increasing the thickness of the measuring electrode 4 makes it difficult to control the cutting location, and the cut surface of the created gap G may become irregular. Therefore, the thickness of the measuring electrode 4 is not limited, but may be, for example, 80 nm or less, 70 nm or less, 60 nm or less, or 50 nm or less. The lower limit of the thickness of the measuring electrode 4 is not particularly limited as long as the tunneling current can be measured, and may be, for example, 2 nm or more, 4 nm or more, 6 nm or more, 8 nm or more, 10 nm or more, 15 nm or more, or 20 nm or more.

[0038] As described above, since the thickness of the measuring electrode 4 is made small and the gap G is formed by the MCBJ, it is preferable that the length of the measuring electrode 4 is greater than the thickness. Although not limited thereto, the length / thickness ratio may be, for example, 10 to 100.

[0039] Incidentally, the device described in Patent Document 1 also has electrodes formed using MCBJ. Therefore, the thickness of the electrodes deposited on the thin film is thin for the above-mentioned reasons. Furthermore, since the device described in Patent Document 1 has nanopores formed in the thin film, the sample moves in the thickness direction of the gap G between the electrodes. On the other hand, when measuring the tunneling current of a sample using the device 1 disclosed in the present application, the sample moves in the length direction of the measurement electrode 4. In other words, the direction of sample movement relative to the gap G is different between the device 1 disclosed in the present application and Patent Document 1. In the device disclosed in the present application, the electric field is applied in the length direction of the measurement electrode 4, so the electric field strength is gentle and the sample movement speed is easy to control. On the other hand, in the device described in Patent Document 1, the electric field is applied in the thickness direction of the electrode (thinner than the length direction of the electrode), so the electric field strength is steep, making it difficult to control the sample movement speed. As described above, the device 1 disclosed in the present application has the advantage of being easier to control the movement speed of the sample passing through the gap G of the measurement electrode 4 compared to the device described in Patent Document 1.

[0040] The length L2 of the sample measurement channel 32 is not particularly limited as long as it is within a range that allows measurement of the tunneling current as the sample passes through. If the length is too long, the entire channel of the device 1 becomes long. On the other hand, if the sample is an elongated sample such as a nucleic acid or peptide (hereinafter referred to as an "elongated sample"), it becomes difficult to maintain the elongated state. While not limited thereto, L2 may be 20 nm or more, 25 nm or more, 30 nm or more, 35 nm or more, 40 nm or more, 45 nm or more, or 50 nm or more. Alternatively, L2 may be 2000 nm or less, 1500 nm or less, 1000 nm or less, 800 nm or less, 600 nm or less, 400 nm or less, 200 nm or less, 180 nm or less, 160 nm or less, 140 nm or less, 120 nm or less, or 100 nm or less. Of course, the length of L2 must be longer than the length of the gap G of the measurement electrode 4.

[0041] To reduce noise caused by the voltage for electrophoresis, it is preferable that the depth H of the flow channel 3 is small. The depth H of the flow channel 3 is not limited, but may be, for example, 200 nm or less, 180 nm or less, 160 nm or less, 140 nm or less, 120 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, or 50 nm or less. On the other hand, the depth H of the flow channel 3 may be, for example, 20 nm or more, 25 nm or more, or 30 nm or more.

[0042] In the device 1 according to the first embodiment, there are no particular limitations on the length (L1 in FIG. 1A) of the first tapered channel 33 and the width (W2 in FIG. 1A) of the sample input channel 33, but it is desirable to make the width of the channel 33 as small as possible. Note that, in order to input the sample liquid, the sample input channel 31 may have a wide portion wider than W2 as needed.

[0043] In the device 1 according to the first embodiment, there are no particular limitations on the width of the sample collection channel 34 or the length (L1a in FIG. 1A) of the second tapered channel 35 that is optionally provided, but it is desirable to make the width of the channel 3 as small as possible. Note that, in order to collect the sample, the sample collection channel 34 may have a wide portion that is wider than W2a as necessary.

[0044] Compared with the devices described in Patent Documents 1 and 2, measuring a tunnel current using the device 1 according to the first embodiment provides the following advantages. (1) The sample contained in the sample liquid introduced into the sample introduction channel 31 is guided by electrophoresis through the first tapered channel 33 to the sample measurement channel 32. Therefore, compared to the nanopore format described in Patent Document 1, even if the amount of sample such as nucleic acid contained in the sample liquid is small, the sample can be more reliably guided to the measurement electrode 4. (2) The nanopore in the device described in Patent Document 1 is a three-dimensional hole formed in a thin film. Therefore, it is very difficult to change the size of the pore within the nanopore. Furthermore, in the device described in Patent Document 1, a chamber is formed so that the sample liquid comes into contact with the thin film. Therefore, it is very difficult to design the opening of the chamber and nanopore without any steps. In other words, there are limitations to the design of the flow channel to facilitate the flow of a sample, such as nucleic acid, contained in the sample liquid into the opening. On the other hand, in Device 1, the flow channel 3 is formed on the substrate 2, so the flow channel 3 can be easily formed in the desired shape by electron beam lithography. (3) By making the absolute value of the width of the sample measurement channel 32 very small and tapering the channel from the sample introduction channel 31 toward the sample measurement channel 32, the cross-sectional area of ​​the channel 3 can be made smaller than in the embodiment described in Patent Document 2. This allows a smaller voltage to be applied to the sample to cause electrophoretic force, thereby reducing noise caused by the voltage for electrophoresis during tunneling current measurement.

[0045] Optional Variations of Device 1 1A to 1C, optional modifications (limitations) of device 1 will be described. Note that the optional modifications of device 1 are embodiments in which each configuration of the embodiment of device 1 is further limited. Therefore, in the optional modifications of device 1, only the limitations will be described, and repeated explanations of matters already described in the first embodiment will be omitted.

[0046] (Variation 1) In device 1, when the width of the connection portion between first tapered channel 33 and sample measurement channel 32 is defined as W1 and the width of the connection portion between first tapered channel 33 and sample introduction channel 31 is defined as W2, the lower limit of W2 / W1 may be 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, and the upper limit may be 50 or less, 40 or less, 30 or less, or 20 or less. Furthermore, when the length of connection portion W1 between first tapered channel 33 and sample measurement channel 32 and the length of the connection portion between the first tapered channel and sample introduction channel are defined as L1, the lower limit of L1 / W2 may be 0.3 or more, 0.4 or more, or 0.5 or more, and the upper limit may be 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, or 5 or less.

[0047] When the shape of the first tapered channel 33 has the above ratio, the following effect is achieved in addition to the effect described in the device 1 according to the first embodiment. (1) By setting the first tapered flow path 33 to the above ratio, it is possible to easily straighten elongated samples in the sample liquid. (2) When the flow path is filled with a solvent and a voltage is applied, electroosmotic flow (EOF) occurs within the flow path, but reverse flow occurs in the area along the wall. By configuring the first tapered flow path 33 within the range described in Modification 1, the generation of EOF is easily suppressed, and the shape of the first tapered flow path 33 provides an acceleration effect by enhancing the electric field. Therefore, it is easy to straighten elongated samples contained in the sample liquid and introduce them into the sample measurement flow path 32.

[0048] (Variation 2) The device 1 may include a second tapered channel 35 disposed between the sample measurement channel 32 and the sample recovery channel 34, the channel width of which increases from the sample measurement channel 32 toward the sample recovery channel 34.

[0049] When the device 1 includes the second tapered flow path 35, in addition to the effects described in the device 1 of the first embodiment and variant example 1, it has the effect of preventing elongated samples from getting stuck at the outlet of the sample measurement flow path 32 and promoting the passage of elongated samples.

[0050] (Variation 3) In addition to Modification 2, the device 1 may satisfy the relationships W1=W1a, W2=W2a, and L1=L1a when the width of the connection portion between the sample measurement channel 32 and the second tapered channel 35 is defined as W1a, the width of the connection portion between the second tapered channel 35 and the sample recovery channel 34 is defined as W2a, and the lengths of the connection portions between the second tapered channel 35 and the sample measurement channel 32 and the second tapered channel 35 and the sample recovery channel 32 are defined as L1a. In other words, the channel 3 may be formed symmetrically with respect to the sample measurement channel 32.

[0051] When the flow path 3 of the device 1 is formed to be symmetrical about the sample measurement flow path 32, in addition to the effects described in the device 1 according to the first embodiment, variant 1 and variant 2, the following effects are achieved. (1) By switching the positive and negative electrodes for electrophoresis, the sample that has passed through the measurement electrode 4 can be measured in the opposite direction. For example, when reading the sequence of nucleic acids or peptides, the same sequence can be confirmed from different directions, which is expected to improve reading accuracy.

[0052] (Other variations) The device 1 disclosed in the present application is not limited to the above-described embodiment 1 and modifications 1 to 3, and may be modified or changed as appropriate within the scope of the technical concept disclosed in the present application. In addition, any component may be omitted in each embodiment.

[0053] For example, the manufactured device 1 may be subjected to a hydrophilization treatment to facilitate the flow of sample liquid. Examples of hydrophilization treatment methods include plasma treatment, surfactant treatment, PVP (polyvinylpyrrolidone) treatment, photocatalytic treatment, and SiO2 film coating. For example, hydroxyl groups can be introduced onto the surface of the device 1 on which the flow channel 3 is formed by plasma treatment for 10 to 30 seconds. The device 1 may also include a cover member 5. Furthermore, electrodes for applying a voltage for electrophoresis may be formed on the sample input flow channel 31 and the sample recovery flow channel 34 of the device 1. The electrodes for electrophoresis will be described later.

[0054] (Embodiment of Tunneling Current Measurement Device) An embodiment of the tunneling current measuring device 100 will be described with reference to Fig. 3. Fig. 3 is a schematic diagram showing an outline of the embodiment of the tunneling current measuring device 100. In addition to the device 1, the tunneling current measuring device 100 includes a power supply for electrophoresis (hereinafter, sometimes referred to as a "first power supply") 6 and a measuring unit 7. The measuring unit 7 includes a tunneling current detecting unit (hereinafter, sometimes referred to as a "detecting unit") 7a and a power supply for tunneling current measurement (hereinafter, sometimes referred to as a "second power supply") 7b.

[0055] 3, a first electrode for electrophoresis (hereinafter sometimes referred to as "first electrode") 61 is formed at a location in contact with the sample liquid in the sample input flow path 31, and a second electrode for electrophoresis (hereinafter sometimes referred to as "second electrode") 62 is formed at a location in contact with the solvent in the sample recovery flow path 34. The first electrode 61 and the second electrode 62 may be components of the device 1 or may be components of the tunneling current measurement apparatus 100.

[0056] The first electrode 61 and the second electrode 62 can be formed of a known conductive metal such as Ag / AgCl, aluminum, copper, platinum, gold, silver, titanium, etc. The first electrode 61 and the second electrode 62 may be formed on the substrate 2, or may be separate from the device 1 and inserted through a hole in the cover member 5.

[0057] FIG. 3 shows an example in which two first power supplies 6, a first power supply 6a connected to the first electrode 61 and a first power supply 6b connected to the second electrode 62, are used to apply a voltage for electrophoresis. In the example shown in FIG. 3, two power supplies are used as the first power supply 6, allowing for independent voltage boosting and voltage drop. Note that the example shown in FIG. 3 is merely illustrative and not limiting. For example, a single first power supply 6 may be used as long as it can apply the electrophoresis voltage described below. The tunneling current measurement device 100 disclosed in the present application can reduce the voltage required for sample electrophoresis by significantly reducing the width of the channel 3 of the device 1, particularly the sample measurement channel 32. Therefore, when measuring the tunneling current generated when a sample passes through the gap between the measurement electrodes 4, the measurement unit 7 can obtain a measured value of the tunneling current with a small noise component. Therefore, the obtained measurement results can be used effectively for applications such as identifying nucleic acid and peptide sequences and analyzing lipids, sugar chains, and synthetic polymers.

[0058] If the voltage applied by the first power supply 6 is too low, the sample movement speed will be slow, requiring a long measurement time. While not limited to this, the voltage may be, for example, 10 mV or more, 15 mV or more, 20 mV or more, 25 mV or more, or 30 mV or more. Meanwhile, the upper limit of the voltage applied by the first power supply 6 may be appropriately set taking into account the accuracy of the analysis unit used to analyze the measurement results and the width of the flow path 3. While not limited to this, the upper limit may be, for example, 5 V or less, 3 V or less, 1 V or less, 500 mV or less, 300 mV or less, 100 mV or less, 90 mV or less, 80 mV or less, 70 mV or less, 60 mV or less, or 50 mV or less. The tunneling current measurement results obtained by the tunneling current measurement device 100 may be analyzed by an analysis unit separate from the tunneling current measurement device 100. Therefore, the analysis unit is not an essential component of the tunneling current measurement device 100.

[0059] The detector 7a of the measurement unit 7 is not particularly limited as long as it has a configuration capable of measuring changes in the tunneling current generated when a sample passes through the gap between the pair of measurement electrodes 4a and 4b, to which a voltage is applied by a tunneling current measurement power supply (hereinafter, sometimes referred to as the "second power supply") 7b. For example, since the changes in the generated tunneling current are on the picoampere level, a known ammeter capable of measuring picoampere-level currents may be used. Alternatively, the current may be calculated from the voltage measured by a voltmeter. The measurement unit 7a may optionally include a current amplifier, a noise eliminator, an A / D converter, etc. If the measurement unit 7a includes a current amplifier, a noise eliminator, an A / D converter, etc., it can provide data that is easy to analyze rather than raw data of the measured tunneling current value. Alternatively, the measurement unit 7a may be configured only to measure changes in the tunneling current, and the current amplifier, noise eliminator, A / D converter, etc. may be included in the analysis unit 8.

[0060] The second power supply 7b applies a voltage to the pair of measurement electrodes 4a and 4b. There are no particular limitations on the voltage applied by the second electrode 7b as long as it allows measurement of the tunneling current. For example, the lower limit may be 20 mV or more, 50 mV or more, or 100 mV or more, and the upper limit may be 750 mV or less, 500 mV or less, or 250 mV or less. The specific configuration of the second power supply 7b is not particularly limited, and any known power supply device may be used.

[0061] By measuring a tunneling current using the tunneling current measurement device 100 disclosed in the present application, it is possible to obtain a measurement result of the tunneling current with less noise components caused by the voltage for electrophoresis.

[0062] (Embodiments of nucleic acid sequence reading device) An embodiment of a nucleic acid sequence reading apparatus 100a will be described with reference to FIG. 4. FIG. 4 is a schematic diagram showing an outline of the embodiment of the nucleic acid sequence reading apparatus 100a. The nucleic acid sequence reading apparatus 100a is the same as the tunneling current measuring apparatus 100, except that in addition to the tunneling current measuring apparatus 100, the nucleic acid sequence reading apparatus 100a includes, as an essential component, an analysis unit 8 that identifies a nucleic acid sequence from the measurement results of the tunneling current acquired by the tunneling current measuring apparatus 100. Therefore, the embodiment of the nucleic acid sequence reading apparatus 100a will be described mainly with reference to the analysis unit 8, and repeated explanations of matters already explained in the embodiment of the tunneling current measuring apparatus 100 will be omitted. Therefore, it goes without saying that matters already explained in the embodiment of the tunneling current measuring apparatus 100 can be adopted even if they are not explicitly explained in the embodiment of the nucleic acid sequence reading apparatus 100a.

[0063] The analysis unit 8 identifies the nucleic acid sequence from the tunneling current measurement results obtained by the tunneling current measurement device 100. More specifically, the analysis unit 8 calculates the conductance from the measured tunneling current. When the tunneling current is measured, the conductance can be calculated by dividing the measured tunneling current by the voltage applied to the pair of measurement electrodes 4a and 4b. The conductance calculated from the tunneling current generated when the nucleic acid passes through the pair of measurement electrodes 4a and 4b varies depending on the type of nucleic acid. Therefore, since the type of nucleic acid can be identified based on the calculated conductance, the nucleic acid sequence can be read by analyzing the measured tunneling current values ​​in a time series.

[0064] Note that the analysis unit 8 may perform the above conductance analysis, and a device separate from the analysis unit 8 may identify specific nucleic acid names such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). Alternatively, the analysis unit 8 may be provided with a memory unit that stores conductances corresponding to the types of nucleic acids, and the analysis unit 8 may directly read the nucleic acid sequence from the tunneling current measurement results by comparing the calculated conductance with the conductance stored in the memory unit. In this specification, "identifying a nucleic acid sequence" includes not only specifically identifying the type of nucleic acid, but also providing conductance information that can identify the type of nucleic acid.

[0065] In this specification, the term "nucleic acid" includes not only the above nucleic acids constituting DNA and RNA, but also artificial nucleic acids. Examples of artificial nucleic acids include, but are not limited to, the following artificial nucleic acids:

[0066] [ka]

[0067] By adding different types of artificial nucleic acids to natural nucleic acids, it is possible to increase the density of nucleic acid memories by increasing the number of bits.

[0068] The nucleic acid sequence reading apparatus 100a may optionally include a display unit 9 for displaying the measured tunneling current value and / or the results of analysis by the analysis unit 8, a program memory 10 in which a program for operating the analysis unit 8 and the display unit 9 has been stored in advance, and a control unit 11 for reading and executing the program stored in the program memory 10. The program may be stored in advance in the program memory 10, or may be recorded on a recording medium and stored in the program memory 10 using an installation means.

[0069] The display unit 9 may be a known display device such as a liquid crystal display, a plasma display, or an organic EL display.

[0070] (Embodiments of tunneling current measurement method and nucleic acid sequence reading method) Next, a tunneling current measurement method using the tunneling current measurement device 100 and a nucleic acid sequence reading method using the nucleic acid sequence reading device 100a will be described with reference to Fig. 5. Fig. 5 is a flowchart of the tunneling current measurement method and the nucleic acid sequence reading method. An embodiment of the tunneling current measurement method includes a sample electrophoresis step (ST1) and a tunneling current measurement step (ST2). An embodiment of the nucleic acid sequence reading method includes a nucleic acid sequence reading step (ST3) in addition to the sample electrophoresis step (ST1) and the tunneling current measurement step (ST2).

[0071] The sample electrophoresis step (ST1) is performed by introducing a sample liquid into the sample introduction channel 31, introducing a solvent into the sample recovery channel 34, and applying a voltage to the first electrode 61 and the second electrode 62. The introduced sample liquid or solvent penetrates the first tapered channel 33, the sample measurement channel 32, and the optional second tapered channel 35 by capillary force, forming a liquid junction. The solvent used to prepare the sample liquid may be electrically conductive. Examples include, but are not limited to, ultrapure water and buffer solutions. Ultrapure water can be produced using, for example, a Milli-Q® Integral 3 (instrument name) manufactured by EMD Millipore (Milli-Q® Integral 33 / 5 / 1015 (catalog number)). Examples of buffer solutions include well-known electrophoresis buffers such as TE buffer and TBE buffer. The buffer concentration is not limited, but may be adjusted appropriately to a range that allows electrophoresis, such as 1 μM or less. Furthermore, in order to reduce the influence of electroosmotic flow (EOF), the sample liquid may contain surfactants such as polyvinyl-pyrrolidone (PVP) as well as amphiphilic chemical substances, if necessary.

[0072] In the tunneling current measurement step (ST2), the tunneling current generated when the sample passes through the gap between the pair of measurement electrodes 4a and 4b arranged in the sample measurement flow path 32 is measured.

[0073] The nucleic acid sequence reading step (ST3) is performed when the sample is a nucleic acid. In the nucleic acid sequence reading step (ST3), the nucleic acid sequence is identified from the tunneling current value obtained in the tunneling current measurement step (ST2) by the method described above (embodiment of the nucleic acid sequence reading device). If necessary, the electrophoresis step (ST1), tunneling current measurement step (ST2), and nucleic acid sequence reading step (ST3) may be performed in advance using a nucleic acid whose sequence is known, and the type of nucleic acid and the calculated conductance may be associated and stored in the memory unit. Furthermore, if the sample is a peptide, nucleic acid may be read as amino acid.

[0074] The following examples are provided to specifically explain the disclosure of the present application, but these examples are provided for reference of specific embodiments and are not intended to limit or restrict the scope of the disclosure of the present application. [Example]

[0075] [Fabrication of Device 1] Example 1 The device was fabricated according to the procedure shown in Figure 2. The specific procedure is as follows. (1) An insulating layer 2b was formed of polyimide on a silicon substrate 2a. (2) A metal layer was deposited on the insulating layer 2b using electron beam lithography and lift-off technology to form the measurement electrode 4. ZEP520A was used as the resist, and gold was used as the material for the metal layer to form the measurement electrode 4. (3) A deposition layer 2c of SiO2 was formed by chemical vapor deposition. A resist layer 2d was laminated on the deposition layer 2c by spin coating. ZEP520A was used as the resist. (4) A pattern of the flow channel 3 including the sample measurement flow channel 32 was formed by electron beam lithography so as to be superimposed on the metal layer for forming the measurement electrode 4 . (5) A flow path 3 was formed by dry etching. After that, a gap (nano-gap G) was formed in the metal layer by bending the substrate 2a using an MCBJ, thereby forming a measurement electrode 4. (6) A cover member 5 made of PDMS, with a sample liquid injection hole and an insertion hole for the electrophoresis electrodes, was fabricated by electron beam lithography. The substrate 2 with the flow path 3 formed therein and the cover member 5 were treated with ozone plasma and bonded together. Ag / AgCl was used for the electrophoresis electrodes, and they were inserted through the holes formed in the cover member 5.

[0076] Fig. 6A is an SEM photograph of the device 1 produced in Example 1, including the measurement electrode 4 and the sample measurement flow path 32 in which the measurement electrode 4 is arranged. Fig. 6B is a photograph of the device 1 with the cover member 5 adhered thereto and the electrophoresis electrode inserted therein.

[0077] The dimensions of the device 1 shown in FIG. 6A will be described with reference to the reference numerals in FIG. 1A. The width W1 of the connection between the first tapered channel 33 and the sample measurement channel 32 was 200 nm, the length L2 of the sample measurement channel 32 was 8 μm, the width W2 of the connection between the first tapered channel 33 and the sample introduction channel 31 was 2 μm, and the length L1 of the first tapered channel 33 was 5 μm, where W1 = W1a, W2 = W2a, and L1 = L1a. The gap G between the pair of measurement electrodes 4a and 4b was adjusted to be between 0.55 nm and 1.0 nm. The depth of the channel 3 was 50 nm.

[0078] <Example 2> By changing the mask used for electron beam lithography, a device was fabricated with a smaller flow channel width than in Example 1 but with the same ratio. The dimensions of the device fabricated in Example 2 were W1 = W1a = 20 nm, W2 = W2a = 200 nm, L1 = L1a = 500 nm, and L2 = 800 nm. The gap G between the pair of measurement electrodes 4a and 4b was 1 nm, and the depth of the flow channel 3 was 20 nm.

[0079] Example 3 [Construction of a nucleic acid sequence reader (tunneling current measurement device)] A battery was used as the electrophoresis power supply 6, and was connected via leads to the electrophoresis electrodes of the device prepared in Example 2. The tunneling current detection unit 7a of the measurement unit 7 used a current / voltage amplification method as an ammeter to measure minute current values ​​as voltages to obtain current values, and a National Instruments digital oscilloscope, an A / D converter, was used as a voltmeter. The current amplifier was made more accurate by incorporating a feedback resistor into a commercially available current amplifier.

[0080] [Method for measuring tunneling current and method for reading nucleic acid sequences] Example 4 (1) Preparation of sample solution The nucleic acid used was λDNA (NIPPON GENE CO., LTD., Tokyo, Japan) with a known sequence. The sample solution was prepared by dissolving the nucleic acid in water. The nucleic acid concentration was 1 μmol / L.

[0081] (2) (Tunnel current measurement (acquisition of nucleic acid information) The sample solution prepared in (1) above was introduced into the sample introduction channel, and the solvent was introduced into the sample recovery channel. DC voltages of 600 mV and -600 mV were applied to the electrophoresis electrodes 61 and 62, respectively. A DC voltage of 100 mV was applied to the measurement electrode 4. The measurement results are shown in Figure 7.

[0082] (3) Reading nucleic acid sequences The conductance was calculated from the waveform of the tunneling current described in (2) above. The nucleic acid sequence was determined from the time axis and the calculated conductance, and it was confirmed to be identical to the sequence of the λDNA used as the sample.

[0083] <Example 5, Comparative Example 1> Tunneling current measurements were performed using the same procedure as in Example 4, except that the DC voltage applied to the electrophoresis electrodes 61 and 62 was changed to 0.1 V (Example 5) and 1000 V (Comparative Example 1). The measurement results are shown in FIG. 8. As shown in FIG. 8A, when an electrophoresis voltage of 1000 V was applied, the difference in the picoampere-level tunneling current generated when the nucleic acid passed through the gap G between the measurement electrodes 4 was completely indistinguishable due to noise. FIG. 8B is a graph enlarging the picoampere-level portion of the tunneling current measurement results in FIG. 8A. As shown in FIG. 8B, when the voltage was 0.1 V, the difference in the picoampere-level tunneling current generated when the nucleic acid passed through the gap G between the measurement electrodes 4 was discernible.

[0084] These results confirmed that increasing the electrophoresis voltage makes it impossible to distinguish differences in the picoampere-level tunneling current generated when nucleic acids pass through the gap G between the measurement electrodes 4 due to noise. As described in Patent Document 2, when a channel formed on a substrate is used to elongate nucleic acids, the channel width and applied voltage should be set to values ​​that allow the nucleic acids to elongate. However, generally, the wider the channel width, the higher the voltage required for the nucleic acids to move through the channel by electrophoresis. Forming fine channels on substrates is known in the field of microfabrication. However, the device disclosed in this application achieves the remarkable effect of being able to read nucleic acid sequences using tunneling current using channels formed on a substrate by optimizing the size and arrangement of the channels.

[0085] [About the migration speed of nucleic acids due to sample solution adjustment] Example 6 Next, when the first tapered channel 33 was formed, an experiment was carried out to confirm the flow of nucleic acid due to electrophoresis. (1) Device fabrication In order to observe the stained nucleic acid under an optical microscope, a device with a larger flow channel than the device prepared in Example 2 was prepared. Note that, since Example 6 was not an experiment to read a nucleic acid sequence, no measurement electrode 4 was formed. Aside from not forming the measurement electrode 4, the device was prepared according to the procedure of Example 1. The dimensions of the device prepared in Example 6 were W1 = W1a = 1 μm, W2 = W2a = 10 μm, L1 = L1a = 20 μm, and L2 = 20 μm. The depth of the flow channel 3 was 20 nm.

[0086] (2) Preparation of sample solution The nucleic acid used was λDNA (NIPPON GENE CO., LTD., Tokyo, Japan). The purchased λDNA was used as is without purification. λDNA was stained with YOYO-1 (registered trademark) Iodide (Thermo Fisher Scientific, Waltham, MA, USA).

[0087] (Sample a: without PVP) The stained λDNA was dissolved in 0.1× Tris-Borateethylenediaminetetraacetic acid (TBE) buffer. (Sample b: with PVP) The stained λDNA was dissolved in 0.05×TBE buffer containing 0.1 w / v% polyvinyl-pyrrolidone (PVP). In all samples, the pH of the TBE buffer was 7.8, and the concentration of λDNA was 0.1 μg / mL.

[0088] (3) Nucleic acid electrophoresis A DC voltage of 5 V was applied to the fabricated device. The migration of nucleic acids was observed using an optical microscope. Figure 9 shows the measurement results. Note that the position (x) on the vertical axis in Figure 9 is the midpoint of the narrowest channel, which corresponds to the sample measurement channel 32. The migration speed of λDNA contained in sample b, which contained PVP, was faster than that of λDNA contained in sample a, which did not contain PVP. This is thought to be because PVP suppressed the electroosmotic flow (EOF) generated in the first tapered channel 33 of device 1. When device 1 disclosed in the present application is used in a tunneling current measurement method or a nucleic acid sequence reading method, it has been revealed that adding a surfactant such as PVP is preferable to improve the nucleic acid reading speed.

[0089] [Regarding the shape of the first tapered flow path 33] Example 7 Next, an experiment was conducted to confirm the effect of the shape of the first tapered channel 33 on the movement speed of the sample. (1) Device fabrication A device was fabricated in which W1 and W1a of the device having the size described in Example 6 (hereinafter referred to as "Device b") were changed as follows. The dimensions other than W1 and W1a were the same as those of Device b.

[0090] Device a: W1 = W1a = 5 μm Device c: W1 = W1a = 0.5 μm

[0091] (2) Preparation of sample solution The sample (beads) used were 40 nm polystyrene florescent particles (FluoSpheres® Carboxylate-Modified Microspheres yellow-green fluorescent, ThermoFisher Co. Ltd., Waltham, MA, USA). The beads were dissolved in 0.1×TBE buffer containing 0.1 w / v% PVP at a concentration of 2 × 10 12 The cells were suspended at a concentration of 1 / mL. (3) Electrophoresis A DC voltage of 5 V was applied to the fabricated device.

[0092] The measurement results are shown in Figure 10. In Figure 10, X represents the portion corresponding to the sample measurement channel 32 (L2 in Figure 1A), and Y represents the portion corresponding to the section from the inlet of the first tapered channel 33 to the outlet of the second tapered channel 35 (L1 + L2 + L1a in Figure 1A) (hereinafter, the portion corresponding to Y may be referred to as the "microchannel portion"). From the measurement results in Figure 10, the analysis shown in Figure 11 was performed. Figure 11(b) is a graph plotting the position and movement speed of individual beads as they pass through Devices a to c. Figure 11(c) is a graph plotting the position and acceleration as individual beads pass through Devices a to c. Figure 11(d) is an enlarged view of Figure 11(b), and Figure 11(e) is an enlarged view of Figure 11(c). Note that since Figures 11(b) to 11(e) are schematic diagrams, all plot symbols are represented by ●.

[0093] The analysis results of Figure 11 and the points that became clear are as follows. (1) The moving speed and acceleration of the beads differed depending on the shape of the first tapered channel 33. (2) As shown in Figures 11(b) and (d), in all of Devices a to c, the migration speed of the beads was stable before and after entering the microchannel, but the migration speed increased after entering the microchannel. Then, the speed peaked at Position 0 and then slowed down toward the exit of the microchannel. The speeds calculated from the trace data of 10 beads (speed at Position (x)) are as follows: Device a: 225±63μm / s Device b: 145±23μm / s Device c: 45±27μm / s Generally, the velocity of a sample particle, v, is determined by the sum of the electrophoretic velocity, vep, and the velocity of the electroosmotic flow, veo, from the channel. The respective velocities, vep and veo, are proportional to the DC electric field, E, and v, and are given by the following equation: v=vep+veo=(μep+μeo)E(1) It was suggested that a stable electric field was formed in the microchannel portion of all of Devices a to c, and it was found that stable flow control was possible. (3) From the data shown in Figure 11(c) and (e), the maximum acceleration in the acceleration region of the microchannel (to the left of Position 0) is as follows: Device a: 52 μm / s 2 Device b: 486 μm / s 2 Device c: 1264 μm / s 2 As is clear from the above calculation results, the larger the value of W2 / W1 (the larger the taper angle of the first tapered channel 33), the greater the acceleration. As the acceleration increases, the force acting on the microchannel increases, contributing to the entropy dissociation energy of elongated samples such as nucleic acids, thereby elongating the elongated samples. Furthermore, as the acceleration increases, the number of samples (number of nucleic acids) passing through the gap G between the measurement electrodes per unit time increases. Furthermore, assuming the same number of samples (number of nucleic acids) passing through the measurement electrodes per unit time, the smaller the W2 / W1, the smaller the value of the electrophoresis voltage, which further reduces measurement noise caused by the electrophoresis voltage. (4) As described above, it was confirmed that adjusting the rate at which the width of the first tapered channel 33 narrows from the sample input channel 31 toward the sample measurement channel 32 can synergistically improve the sample reading speed and / or reduce measurement noise caused by the electrophoresis voltage, and elongate elongated samples such as nucleic acids. [Industrial Applicability]

[0094] The device disclosed in the present application can reduce noise caused by the electrophoresis voltage when measuring the tunneling current generated when a sample passes through the measurement electrode 4. Therefore, the device is useful for the development of analytical equipment in the analytical instrument industry. [Explanation of symbols]

[0095] 1...device, 2...base material, 2a...substrate, 2b...insulating layer, 2c...deposition layer, 2d...resist layer, 3...flow path, 31...sample input flow path, 32...sample measurement flow path, 33...first tapered flow path, 34...sample recovery flow path, 35...second tapered flow path, 4, 4a, 4b...measurement electrodes, 5...cover member, 6, 6a, 6b...power supply for electrophoresis, 61...first electrode for electrophoresis, 62...second electrode for electrophoresis, 7...measuring unit, 7a...tunneling current detecting unit, 7b...power supply for tunneling current measurement, 8...analysis unit, 9...display unit, 10...program memory, 11...control unit, 100...tunneling current measuring device, 100a...nucleic acid sequence reading device

Claims

1. 1. A device for use in measuring tunneling current, the device comprising: A substrate; a channel formed in a substrate; a pair of measurement electrodes for measuring the tunneling current when the sample passes through; Including, The flow path is a sample input channel; a sample measurement flow channel in which a measurement electrode is disposed; a first tapered channel disposed between the sample introduction channel and the sample measurement channel, the channel width of which narrows from the sample introduction channel toward the sample measurement channel (excluding the change in depth of the first tapered channel from the sample introduction channel toward the sample measurement channel); a sample collection flow path for collecting the sample that has passed through the sample measurement flow path; Including, The width of the connection portion between the first tapered channel and the sample measurement channel is 20 nm to 200 nm, When the width of the connection portion between the first tapered channel and the sample measurement channel is defined as W1 and the width of the connection portion between the first tapered channel and the sample introduction channel is defined as W2, W2 / W1 is 10 to 50, When the length of the connecting portion between the first tapered channel and the sample measurement channel and the connecting portion between the first tapered channel and the sample introduction channel is defined as L1, L1 / W2 is 0.3 to 10. device.

2. L1 / W2 is 0.5 to 5 The device of claim 1 .

3. The length of the sample measurement channel is 20 nm to 1000 nm.

3. A device according to claim 1 or 2.

4. a second tapered channel disposed between the sample measurement channel and the sample recovery channel, the channel width of which increases from the sample measurement channel toward the sample recovery channel; A device according to any one of claims 1 to 3.

5. The width of the connection portion between the sample measurement channel and the second tapered channel is defined as W1a, The width of the connection portion between the second tapered channel and the sample recovery channel is defined as W2a, When the length of the connection part between the second tapered channel and the sample measurement channel and the connection part between the second tapered channel and the sample recovery channel are defined as L1a, The relationships W1=W1a, W2=W2a, and L1=L1a are satisfied. A device according to any one of claims 1 to 4.

6. A device comprising: the device according to any one of claims 1 to 5; a power supply for electrophoresis; and a measurement unit; The electrophoresis power supply applies a voltage of 10 mV to 5 V to the electrophoresis electrodes. Tunnel current measurement device.

7. a tunneling current measuring device according to claim 6 and an analyzing unit, the sample is a nucleic acid, The analysis unit identifies the nucleic acid sequence from the tunneling current measurement results obtained by the tunneling current measurement device. Nucleic acid sequence reading device.

8. A tunneling current measurement method using a device, The device is A substrate; a channel formed in a substrate; a pair of measurement electrodes for measuring the tunneling current when the sample passes through; Including, The flow path is a sample input channel; a sample measurement flow channel in which a measurement electrode is disposed; a first tapered channel disposed between the sample introduction channel and the sample measurement channel, the channel width of which narrows from the sample introduction channel toward the sample measurement channel (excluding the change in depth of the first tapered channel from the sample introduction channel toward the sample measurement channel); a sample collection flow path for collecting the sample that has passed through the sample measurement flow path; Including, The width of the connection portion between the first tapered channel and the sample measurement channel is 20 nm to 200 nm, When the width of the connection portion between the first tapered channel and the sample measurement channel is defined as W1 and the width of the connection portion between the first tapered channel and the sample introduction channel is defined as W2, W2 / W1 is 10 to 50, When the length of the connection portion between the first tapered channel and the sample measurement channel and the length of the connection portion between the first tapered channel and the sample introduction channel are defined as L1, L1 / W2 is 0.3 to 10; The tunnel current measurement method is as follows: a sample electrophoresis step of electrophoresing the sample in the sample input channel toward the sample recovery channel by applying a voltage to the sample input channel and the sample recovery channel; a measurement step of measuring a tunnel current when a sample passes through a gap between a pair of measurement electrodes arranged in a sample measurement flow path; A tunneling current measurement method comprising:

9. In the sample electrophoresis step, a voltage of 10 mV to 5 V is applied. The tunneling current measuring method according to claim 8.

10. L1 / W2 is 0.5 to 5 10. The tunnel current measuring method according to claim 8 or 9.

11. the sample is a nucleic acid, a nucleic acid sequence reading step for identifying a nucleic acid sequence from the tunneling current measurement result obtained by the measurement step of the tunneling current measurement method according to any one of claims 8 to 10; A method for reading a nucleic acid sequence, comprising:

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