WO2020138021A1

WO2020138021A1 - Sample identification method, device for identifying sample, and sample identification apparatus

Info

Publication number: WO2020138021A1
Application number: PCT/JP2019/050469
Authority: WO
Inventors: 真楠筒井; 彰秀有馬; 正輝谷口; 一道横田; 川合　知二
Original assignee: 国立大学法人大阪大学
Priority date: 2018-12-25
Filing date: 2019-12-24
Publication date: 2020-07-02
Also published as: JPWO2020138021A1; JP7398118B2

Abstract

The purpose of the present invention is to provide a sample identification method, a device for identifying a sample, and a sample identification apparatus that improve identification accuracy when a sample is identified using a device in which two or more through-holes through which the sample passes are formed in a substrate. The problem can be solved by a method, which identifies a sample (S1, S2) in a sample liquid, and comprises at least: a sample passing step of causing the sample to pass through a through-hole (3) formed in a substrate (2); an ion current measuring step of measuring a change in an ion current when the sample passes through the through-hole; and a sample identification step of identifying the sample in the sample liquid from the measured value of the ion current, wherein two or more through-holes are formed in the substrate, and the through-holes are arranged such that the distance between the adjacent through-holes is at least 200 nm or more.

Description

Sample identification method, sample identification device, and sample identification device

The disclosure in the present specification relates to a sample identification method, a sample identification device, and a sample identification device.

BACKGROUND ART A device that forms a through-hole (nanopore) in a substrate and measures an ionic current when a sample passes through the through-hole is attracting attention as a device that can be widely applied to sensing bacteria, viruses, DNA, proteins and the like. ..

As a related technique, for example, it is possible to identify the volume of a sample by detecting a change in ion current that occurs when the sample passes through pores (micropores) formed on a substrate such as silicon (Non-Patent Document 1). It is known that the ion current corresponding to the shape of the sample can be measured by making the thickness of the through hole thinner than that of the sample (see Patent Document 1). It is also known to form two or more through holes on a substrate (see Non-Patent Document 2).

JP, 2015-39365, A

As described in Non-Patent Document 2 above, it is known to form two or more through holes on a substrate as a device for measuring an ion current. Then, as shown in the ABSTRACT diagram of Non-Patent Document 2, in order to improve the sample capturing efficiency per unit time as the number of through holes formed in the substrate increases, the distance between the through holes should be shortened. Is disclosed as desirable.

However, the inventors of the present invention conducted further research using a device in which two or more through holes were formed in the substrate. (1) When identifying the sample to be analyzed, if the distance of the through holes was shortened, the measurement was performed. It is difficult to make a difference in the peak value of the generated ionic current, and as a result, the identification accuracy of the sample decreases, and in particular, it becomes difficult to identify the samples of different sizes. (2) The longer the distance of the through hole, the shorter It was newly found that, contrary to the case, the peak value of the measured ionic current is likely to have a difference and the identification accuracy of the sample is increased.

That is, an object of the disclosure in the present specification is to provide a sample identification method, a sample identification device, and a sample identification device that can improve identification accuracy when identifying a sample using a device in which two or more through-holes through which a sample passes are formed in a substrate. , To provide a sample identification device.

The disclosure in the present specification relates to a sample identification method, a sample identification device, and a sample identification device described below.

(1) A method for identifying a sample in a sample liquid, comprising:
The identification method is
A sample passing step of passing the sample through a through hole formed in the substrate;
An ion current measuring step of measuring a change in ion current when the sample passes through the through hole,
From the value of the measured ion current, a sample identification step of identifying the sample in the sample liquid,
Including at least
Two or more through holes are formed in the substrate, and the through holes are arranged such that the distance between adjacent through holes is at least 200 nm or more.
Sample identification method.
(2) The distance between the adjacent through holes is 1 μm or more,
The sample identification method according to (1) above.
(3) The distance between the adjacent through holes is longer than 10 μm,
The sample identification method according to (2) above.
(4) The sample liquid contains samples of different sizes,
The sample identification method according to any one of (1) to (3) above.
(5) A device used for identifying a sample in a sample solution,
The device is
Board,
Two or more through holes formed in the substrate,
Including,
The two or more through holes are arranged such that the distance between adjacent through holes is at least 200 nm or more.
Device for sample identification.
(6) The distance between the adjacent through holes is 1 μm or more,
The sample identification device according to (5) above.
(7) The distance between the adjacent through holes is longer than 10 μm,
The sample identification device according to (6) above.
(8) A first chamber member that forms a first chamber filled with an electrolytic solution by a surface including at least a through hole on the first surface side of the substrate,
A second chamber member that forms a second chamber filled with an electrolytic solution by at least the surface including the through hole on the second surface side of the substrate;
The sample identification device according to any one of (5) to (7) above, including:
(9) The sample identification device according to (8) above,
A first electrode formed in the first chamber;
A second electrode formed in the second chamber;
An ammeter for measuring an ion current when a sample passes through the through hole,
including,
Sample identification device.

By implementing the sample identification method using the device for sample identification disclosed in the present invention, the identification accuracy of the sample is improved, and even samples of different sizes can be identified.

FIG. 1 is a schematic view of a device 1a according to the first embodiment, FIG. 1A is a top view of the device 1a, and FIGS. 1B and 1C are cross-sectional views taken along line YY′ of FIG. 1A. FIG. 2 is a schematic cross-sectional view of the device 1b according to the second embodiment. FIG. 3 is a schematic cross-sectional view of an embodiment of the identification device 100. FIG. 4 is a flowchart of the sample identification method. 5A to 5E are charts showing the measurement results of the ion current in Example 1 of the sample identification method. 6A to 6E are charts showing the measurement results of the ion current in Example 2 of the sample identification method. 7A to 7E are charts showing the measurement results of the ion current in Example 3 of the sample identifying method. 8A to 8H are charts showing measurement results of ion currents in Example 4 of the sample identifying method. 9A and 9B are charts showing measurement results of ion currents in Example 5 of the sample identification method. 10A to 10C are charts showing measurement results of ion current in Example 6 of the sample identification method. The upper part of FIGS. 10A to 10C is a graph showing the change in the base current when measuring the ion current, and the lower part is a graph showing the peak value of the ion current when each sample passes through the through-hole 3 as dots. ..

Hereinafter, a sample identification method (hereinafter, may be simply referred to as “identification method”), a device for sample identification (hereinafter, may be simply referred to as “device”), and a sample identification device (hereinafter, simply referred to as “device”). , May be simply referred to as “identification device”).

(First Embodiment of Device)
A device 1a according to the first embodiment will be described with reference to FIGS. 1A to 1C. 1A is a top view of the device 1a, and FIGS. 1B and 1C are cross-sectional views taken along the line YY′ of FIG. 1A. The device 1a includes at least the substrates 2 and the through holes 3 of two or more. The through hole 3 is formed so as to penetrate the substrate 2 in the direction from the first surface 21 of the substrate 2 to the second surface 22 opposite to the first surface 21.

The substrate 2 is not particularly limited as long as it is an insulating material generally used in the field of semiconductor manufacturing technology. For example, Si, Ge, Se, Te, GaAs, GaP, GaN, InSb, InP, SiN, etc. may be mentioned. The substrate 2 is made of a material such as SiN, SiO ₂ , HfO ₂ or the like, which is a thin film called a solid membrane, or is made of graphene, graphene oxide, molybdenum dioxide (MoS ₂ ), boron nitride (BN) or the like. It may be formed into a sheet shape called a two-dimensional material. Since the sample detection sensitivity increases as the volume of the through hole 3 decreases, it is preferable that the substrate 2 forming the through hole 3 be thin. For example, it is preferably 5 μm or less, more preferably 500 nm or less, further preferably 100 nm or less, particularly preferably 50 nm or less. Note that, for example, graphene can be used to manufacture the substrate 2 having a film thickness of 1 nm or less. When a solid membrane or a two-dimensional material is used as the substrate 2, the film thickness can be made extremely thin. However, if the film thickness of the substrate 2 is very thin, it may be difficult to handle it without damage. Therefore, the substrate 2 may have a laminated structure in which a solid membrane or a two-dimensional material is laminated on the supporting plate formed of the above-mentioned insulating material. In the case of a laminated structure, a solid membrane or a two-dimensional material may be laminated on a support plate having holes larger than the through holes 3 and the through holes 3 may be formed in the solid membrane or the two-dimensional material.

The through hole 3 is formed so as to penetrate the substrate 2 in the direction from the first surface 21 of the substrate 2 to the second surface 22 which is the surface opposite to the first surface 21. When detecting the ion current, the smaller the volume of the through hole 3, the higher the sensitivity. Therefore, while making the substrate 2 thin, the size of the through hole 3 may be appropriately adjusted so that it is larger than the sample to be measured, but not too large. When the cross-sectional shape parallel to the first surface 21 of the through hole 3 is circular, the size of the through hole 3 means the diameter. When the cross-sectional shape parallel to the first surface 21 of the through hole 3 is not circular, the size of the through hole 3 means the diameter of the circumscribed circle of the cross section. The through holes 3 may be formed by etching or the like, as shown in the examples described later. Further, as shown in FIG. 1B, the through hole 3 has the same shape as the first opening 31 of the through hole 3 on the first surface 21 side and the second opening 32 of the through hole 3 on the second surface 22 side. It may be formed in. Alternatively, as shown in FIG. 1C, the sizes of the first opening 31 and the second opening 32 are different, in other words, the through hole 3 extends from the first surface 21 toward the second surface 22 in the base material 2. It may be formed so as to spread.

The device 1a according to the first embodiment has two or more through-holes 3 formed on the substrate 2, but is arranged so that the distance d1 between adjacent through-holes 3 is at least 200 nm or more. Is. In the present specification, the “distance between adjacent through holes” is a line connecting the outer edge of any through hole 3 and the outer edge of any adjacent through hole 3 (FIG. 1A), as shown in FIG. 1A. Means the shortest distance (d') among the dashed lines d'and d'' with an arrow. Moreover, when there are a plurality of adjacent through holes 3 and the distances connecting the outer edges of the through holes 3 are different (d1, d2 in FIG. 1A), the “distance between adjacent through holes” is the shortest. It means a distance (d1). However, as shown in FIG. 1C, when the size of the first opening 31 of the first surface 21 and the size of the second opening 32 of the second surface 22 are different, the “distance between adjacent through holes” is larger. Means the distance connecting the outer edge of the opening (first opening 31 in FIG. 1C). As shown in Examples and Comparative Examples to be described later, when the distance between the through holes 3 (openings) is shortened, the peak value of the measured ionic current becomes low, making it difficult to identify the sample. Therefore, the distance between adjacent through holes is 200 nm or more, 300 nm or more, 400 nm or more, 600 nm or more, 1 μm or more, 2 μm or more, 5 μm or more, 10 μm or more, 15 μm or more, 20 μm or more, etc., depending on the desired identification accuracy. Adjust it. On the other hand, the upper limit of the distance between adjacent through-holes is not particularly limited from the viewpoint of sample identification, but if the distance is too long, the number of through-holes 3 per unit area of the substrate 2 will decrease and the measurement efficiency will increase. descend. Therefore, it may be set as appropriate in consideration of the area of the substrate 2 and the desired measurement efficiency.

As described above, if the through holes 3 formed on the substrate 2 are arranged such that the distance between the arbitrary through hole 3 and the through hole 3 closest to the through hole 3 is 200 nm or more, There is no particular limitation. For example, the through holes 3 may be arranged at random, or may be arranged so as to have a predetermined shape such as a square shown in FIG. 1A. Alternatively, as in the close-packed shape, the through holes may be arranged such that the distances between adjacent through holes are all the same.

The sample is not particularly limited as long as it has a volume, and examples thereof include biological substances such as bacteria, cells, viruses, DNA, RNA, proteins, pollen, sulfur oxides (SOx), nitrogen oxides (NOx), Examples thereof include volatile organic compounds (VOC) and oxide minerals (non-biological substances such as silicon, aluminum, titanium and iron).

(Second Embodiment of Device)
A second embodiment of the device will be described with reference to FIG. FIG. 2 is a schematic cross-sectional view of the device 1b according to the second embodiment. The device 1b shown in FIG. 2 includes a first chamber member 51 capable of forming a first chamber 5 filled with an electrolytic solution together with a surface of the first surface 21 of the substrate 2 including the first opening 31 of the through hole 3, and a substrate. At least a second chamber member 61 capable of forming the second chamber 6 filled with the electrolytic solution is formed by the second second surface 22 and the surface including the second opening 32 of the through hole 3.

The first chamber member 51 and the second chamber member 61 are preferably formed of an electrically and chemically inert material, for example, glass, sapphire, ceramic, resin, rubber, elastomer, SiO ₂ , SiN, Al. ₂ O _{3 and the} like.

The first chamber 5 and the second chamber 6 are formed so as to sandwich the through hole 3, and the sample introduced into the first chamber 5 can be moved to the second chamber 6 through the through hole 3. There is no particular limitation. For example, the first chamber member 51 and the second chamber member 61 may be separately prepared and bonded to the substrate 2 so as to be liquid-tight. Alternatively, one surface may form a substantially rectangular parallelepiped box member, the substrate 2 may be inserted and fixed in the center of the box, and then the open surface may be liquid-tightly sealed. In that case, the first chamber member 51 and the second chamber member 61 do not mean separate members but a part of a box member divided by the substrate 2. Although not shown, holes are formed in the first chamber member 51 and the second chamber member 61 to fill and discharge the electrolyte solution and the sample solution, and to insert electrodes and/or leads as needed. You may.

(Embodiment of identification device)
An embodiment of the identification device will be described with reference to FIG. FIG. 3 is a schematic cross-sectional view of an embodiment of the identification device 100. The identification device 100 includes, in addition to the device 1b, a first electrode 52 formed in a portion in contact with the electrolytic solution in the first chamber 5, a second electrode 62 formed in a portion in contact with the electrolytic solution in the second chamber 6, At least an ammeter 7 for measuring the ion current when the samples S1 and S2 pass through the through hole 3 is included.

Further, the identification device 100, if necessary, the analysis unit 8 that analyzes the ion current measured by the ammeter 7, the display unit 9 that displays the measured ion current value and/or the result analyzed by the analysis unit 8. A program memory 10 in which a program for causing the analysis unit 8 and the display unit 9 to function is stored in advance, and a control unit 11 for reading and executing the program stored in the program memory 10 may be included. The program may be stored in the program memory 10 in advance, or may be recorded in a recording medium and stored in the program memory 10 using an installation unit.

The first electrode 52 and the second electrode 62 can be formed of a known conductive metal such as aluminum, copper, platinum, gold, silver and titanium. The first electrode 52 and the second electrode 62 are formed so as to sandwich the through hole 3, and a DC current is applied to transport the ions in the electrolytic solution. Therefore, the first electrode 52 has only to be formed at a position in contact with the electrolytic solution in the first chamber 5, and may be formed on the surface of the substrate 2, the inner surface of the first chamber member 51, or the space in the first chamber 5. It may be arranged via the lead 53. Similarly to the first electrode 51, the second electrode 62 may be formed at a position in contact with the electrolytic solution in the second chamber 6, on the surface of the substrate 2, the inner surface of the second chamber member 61, or the second chamber. It may be arranged in the space in 6 via the lead 63. In the example shown in FIG. 3, the first electrode 52 is formed on the inner surface of the first chamber member 51, and the second electrode 62 is formed on the inner surface of the second chamber member 61. The electrode 62 may be inserted through the holes formed in the first chamber member 51 and the second chamber member 61.

The first electrode 52 is connected to the power supply 54 and the earth 55 via the lead 53. The second electrode 62 is connected to the ammeter 7 and the ground 64 via the lead 63. Although the power supply 54 is connected to the first electrode 52 side and the ammeter 7 is connected to the second electrode 62 side in the example shown in FIG. 3, the power supply 54 and the ammeter 7 may be provided on the same electrode side. Good.

The power supply 54 is not particularly limited as long as it can supply a direct current to the first electrode 52 and the second electrode 62. The ammeter 7 is not particularly limited as long as it can measure the ion current generated when the first electrode 52 and the second electrode 62 are energized with time. Although not shown in FIG. 3, a noise removing circuit, a voltage stabilizing circuit, or the like may be provided if necessary.

When the sample passes through the through hole 3 of the identification device 100, the ion current flowing through the through hole 3 is blocked by the sample, and the ion current decreases. The amount of decrease in the ion current is proportional to the volume of the sample in the through hole 3. However, if the distance of the through hole 3 (opening) is too short, the identification accuracy is lowered, and as shown in FIG. 3, even when the sizes of the samples S1 and S2 are different, there is a difference in the peak value of the measured ion current. Will be difficult to remove. On the other hand, in the identification device 100 according to the embodiment, the through holes 3 (openings) adjacent to each other are arranged such that the distance between them is 200 nm or more. Therefore, there is a difference in the peak value of the measured ionic current, so that the sample can be identified according to the size of the samples S1 and S2 passing through the through hole 3. The smaller the volume of the through hole 3 is, the higher the measurement sensitivity of the ion current is. Therefore, the thickness of the substrate 2 (the distance between the first surface 21 and the second surface 22) is preferably thin. Although it depends on the sample concentration (number) in the sample liquid and the number of through holes 3 formed on the substrate 2, the probability that the sample will pass through the through holes 3 at the same time is extremely low. Therefore, even if several samples pass through the through-hole 3 at the same time, they are only recognized as specific peaks and have no effect on identifying what kind of sample is contained in the sample solution. Does not give.

The analysis unit 8 analyzes the value (peak value) of the ion current measured by the ammeter 7. As described above, the value of the ionic current changes depending on the size of the sample. Therefore, the sample can be identified by performing data analysis by the analysis unit 8 based on the measured ion current value.

The display unit 9 only needs to be able to display the value (peak value) of the measured ion current and the result analyzed by the analysis unit 8, and a known display device such as a liquid crystal display, a plasma display, an organic EL display may be used.

(Embodiment of sample identification method)
Next, a sample identification method using the identification device 100 will be described with reference to FIG. FIG. 4 is a flowchart of the sample identification method. The embodiment of the sample identifying method includes a sample passing step (ST1), an ion current measuring step (ST2), and a sample identifying step (ST3).

In the sample passing step (ST1), the sample is passed through the through hole 3 formed in the substrate 2. A sample preparation step may be included before the sample passing step. The sample preparation step can be performed by the following procedure.
(1) The first chamber 5 and the second chamber 6 are filled with an electrolytic solution. The electrolyte is not particularly limited as long as the first electrode 52 and the second electrode 62 can be energized, and TE buffer, PBS buffer, HEPES buffer, KCl aqueous solution or the like may be used. At this time, a liquid junction is established between the inside of the first chamber 5 and the inside of the second chamber 6 via the through hole 3.
(2) Add the sample to the first chamber 5.
Although the above (1) and (2) may be performed separately, the electrolytic solution containing the sample may be filled in the first or second chamber.

The sample passing step (ST1) can be performed by energizing the first electrode 52 and the second electrode 62 arranged in the first chamber 5 and the second chamber 6 filled with the electrolytic solution in the preparation step. Samples such as bacteria have surface electrification. Therefore, when the first electrode 52 and the second electrode 62 are energized, in addition to normal diffusion, the sample added to the first chamber 5 passes through the through-hole 3 formed in the substrate 2 by electrophoresis, and then enters the second chamber 6. Although moving, the pressure may be applied to the solution in which the sample is dispersed by a pump or the like as necessary, and the sample may pass through the through hole 3 by a water flow.

In the ion current measurement step (ST2), the value of the ion current generated by energization is measured with the ammeter 7 over time. Then, in the sample identifying step (ST3), the sample may be identified from the peak value of the ion current measured in the ion current measuring step (ST2). Specifically, the size of the sample can be identified by the degree of decrease in the measured value of the ionic current.

The present invention will be specifically described below with reference to examples, but the examples are provided merely for the purpose of explaining the present invention and for reference of specific modes thereof. These exemplifications are intended to illustrate certain specific embodiments of the invention, but are not meant to limit or limit the scope of the invention disclosed herein.

[Fabrication of device]
First, a silicon wafer (E&M CO., LTD) with a plane orientation (100) having a silicon nitride film of 50 nm on both surfaces was cut into 25 mm squares. A 500 μm square in which holes are formed by an RIE device (RIE-10NR, SAMCO CO., Ltd.) is covered with an etching prevention metal mask in which holes of approximately 500 μm square are formed on one surface of the substrate. The silicon nitride film was removed only in the area (1) to expose the silicon surface. After that, only the exposed portion of the silicon is selectively exposed to a hot plate (Hot plate NINOS ND-1, As One CO., Ltd.) at 125° C. for about 3 hours by using an aqueous potassium hydroxide solution (Wako Pure Chemical Industries, Ltd.). ) Wet etching was performed above. By this operation, silicon was etched until the silicon nitride film on the other surface of the substrate was reached. The silicon holes reaching the silicon nitride film were about 150 μm square.

Next, the pattern of the through holes 3 was drawn by an electron beam drawing method at approximately the center of the silicon nitride film covering the above-described about 150 μm square silicon holes. Then, it was immersed in an image solution for development, and reactive etching was performed by an RIE apparatus to form a cylindrical through hole 3 in the silicon nitride film. The device was produced by combining various embodiments such that the number of through holes 3 was 1 to 7, the diameter was 300 nm to 3.4 μm, and the distance between adjacent through holes was 30 nm to 10 μm. The length of the through hole 3 was about 50 nm.

[Production of identification device]
Next, a polymer block (manufactured by TORAY) made of dimethylpolysiloxane (PDMS) having electrodes, an electrolytic solution, and holes for introducing a sample is liquid-tightly attached to the upper and lower sides of the substrate 2 prepared in [Production of device]. Then, the first chamber 5 and the second chamber 6 were produced. The volumes of the first chamber 5 and the second chamber 6 were each about 10 μl. A silver-silver chloride electrode was used for the first electrode 52 and the second electrode 62, and the electrodes were inserted into the first chamber 5 and the second chamber 6 through the holes provided in the polymer block. A battery-driven bias power source (Axis Net) was used as the power source 54, and was connected to the first electrode 52 via a lead. The ammeter 7 uses a current amplifier and a digitizer (NI 5922, National Instruments) having a high time resolution of 1 MHz to acquire data, and the acquired data is a RAID drive HDD (HDD-8263, National Instruments Co.). Stored in.

[Example 1 of sample identification method]
1. Preparation of sample solution As a sample, carboxy group-modified polystyrene particles (Reagent Microspheres manufactured by Thermo Scientific) having a diameter of 780 nm were used, and polystyrene particles were suspended in 1 ml of an electrolytic solution (TE buffer: TE (pH 8.0) manufactured by Nippon Gene Co., Ltd.). A sample liquid was prepared by suspending 1 μl of the liquid.

2. Measurement of Ion Current The second chamber 6 of the produced identification device was filled with an electrolytic solution (TE buffer: TE (pH 8.0) manufactured by Nippon Gene Co., Ltd.). Next, the above 1. The sample liquid prepared in step 1 was added to the first chamber 5, a voltage of 800 mV was applied to the first electrode 52 and the second electrode 62, and the ion current I _ion was measured.

5A to 5E are charts showing measurement results of ion current. The location where the ionic current value is significantly reduced indicates that the polystyrene particles have passed through the through holes 3. 5B to 5D use a device in which four through holes 3 having a diameter of 1.2 μm are arranged in a substantially square shape, and the distance of the through holes 3 is 100 nm in FIG. 5B and 1 μm in FIG. 5C. 5D was arranged so as to be 10 μm. 5A and 5E show an example using a device for comparison, and FIG. 5A shows a through hole having a diameter of 2.4 μm, which is the same as the size of four through holes having a diameter of 1.2 μm. One 3 was arranged. Further, in FIG. 5E, one through hole 3 having a diameter of 1.2 μm, which is the same as in FIGS. 5B to 5D, is arranged.

As is clear from FIGS. 5B to 5D, the shorter the distance between the adjacent through holes 3 is, the smaller the peak value of the ionic current is, although the through holes 3 have the same size. Then, referring to FIGS. 5A and 5B, although four through holes 3 are arranged in FIG. 5B, since the distance between adjacent through holes 3 is as short as 100 nm, the peak value of the measured ion current is as shown in FIG. 5B. The value was close to the peak value of the ion current in FIG. 5A in which one through hole 3 having a size obtained by adding the four through holes was formed. On the other hand, referring to FIGS. 5D and 5E, although four through holes 3 are arranged in FIG. 5D, since the distance between adjacent through holes 3 is 10 μm, the measured peak value of the ion current is It was almost the same as the peak value of the ion current in FIG. 5E in which one through hole 3 having the same size was formed.

[Example 2 of sample identification method]
Next, an experiment was performed using polystyrene particles having a diameter of 900 nm as a sample and a device in which seven through holes 3 were arranged. 6A to 6E are charts showing measurement results of ion current. In FIGS. 6B to 6D, seven through holes 3 having a diameter of 1.2 μm are arranged, and the distances between all the adjacent through holes 3 are 100 nm in FIG. 6B, 1 μm in FIG. 6C, and 10 μm in FIG. 6D. So that Further, FIGS. 6A and 6E show an example using a device for comparison, and FIG. 6A shows one through hole 3 having a diameter of 3.2 μm, which has the same area as seven through holes having a diameter of 1.2 μm. I placed it. Further, in FIG. 6E, one through hole 3 having a diameter of 1.2 μm, which is the same as in FIGS. 6B to 6D, is arranged. The ionic current was measured by the same procedure as in [Example 1 of sample identification method] except the size of the sample and the arrangement of the through holes 3. Also in the following examples, the procedure for measuring the ion current is the same except the arrangement of the sample and the device.

As is apparent from FIGS. 6A to 6E, even when the number of through holes 3 was changed from 4 to 7, the peak value of the ion current showed the same tendency as in FIGS. 5A to 5E. However, as is clear from FIGS. 6A and 6B, the peak value of the ion current when the seven through holes 3 were arranged at a distance of 100 nm was almost the same as that in FIG. 6A. In other words, even though seven through holes 3 were formed, the measurement accuracy was the same as when one through hole having the same opening area was formed. Further, as is clear from FIGS. 6D and 6E, the peak value of the ionic current when the seven through holes 3 are arranged at a distance of 10 μm is slightly lower than the peak value of the ionic current of FIG. 6E. ..

[Example 3 of sample identification method]
Next, an experiment was conducted using a device in which polystyrene particles having a diameter of 200 nm were used as a sample, the diameter of the through holes 3 was 300 nm, the number of the through holes 3 was 1 to 7, and the distance between the through holes 3 was 100 nm. I went. 7A to 7E are charts showing measurement results of ion current. As is clear from FIGS. 7A to 7E, the larger the number of through holes 3, the smaller the peak value of the ion current. On the other hand, it was confirmed that the larger the number of through holes 3, the larger the number of peaks per unit time, in other words, the number of samples passing through the through holes 3 per unit time.

From the above results, as the number of the through holes 3 formed on the substrate 2 is increased, the peak value of the ion current becomes larger as the distance between the adjacent through holes 3 becomes longer, so that the identification accuracy (measurement sensitivity) is increased. It was clarified that the number of samples passing through the through-hole 3 per unit time increased as the number of the through-holes 3 increased. Therefore, the number and the distance of the through holes 3 formed per unit area may be appropriately adjusted in consideration of the number of the through holes 3 formed and the identification accuracy.

[Example 4 of sample identification method]
Next, an experiment was conducted to examine the distance of the through hole 3 in more detail. Polystyrene particles having a diameter of 780 nm are used as a sample, two through holes 3 having a diameter of 1.2 μm are arranged, and the distances of the through holes 3 are 30 nm (FIG. 8B), 50 nm (FIG. 8C), 100 nm (FIG. 8D), 200 nm. (FIG. 8E), 400 nm (FIG. 8F), 600 nm (FIG. 8G), 1000 nm (FIG. 8H). As a comparison target, an experiment was also performed with a device (FIG. 8A) in which one through hole 3 having a diameter of 1.44 μm was arranged. 8A to 8H are graphs in which the measured values of the ionic current when a plurality of samples pass through the through hole 3 are superimposed, the horizontal axis represents the peak value of the ionic current, and the vertical axis represents the corresponding ionic current. Represents the number of samples showing the peak value of.

As shown in FIG. 8A, when the size of the through hole 3 is relatively large compared to the sample size, the distribution of the peak value of the ionic current is largely divided into two. This is because, when the sample passes through the through hole 3, the non-uniformity of the electric field generated at that moment is different when the sample passes through the vicinity of the center of the through hole 3 and when the sample passes through the peripheral portion of the through hole 3. It is considered that the ion current distribution was observed depending on the position where the particles passed through the pores, because the ion current decreased a little when passing through the central part as compared with when passing through the peripheral part. On the other hand, as shown in FIG. 8B, when the distance between the through holes 3 is as short as 30 nm, the distribution of the peak value of the ion current is largely divided into two, as in FIG. 8A. As is apparent, the difference between the two different distributions of the peak value of the ion current becomes smaller as the distance of the through hole 3 becomes longer. Then, as shown in FIG. 8E, when the distance between the through holes 3 is set to 200 nm, two distinct distributions of the peak value of the ion current cannot be seen, and in addition, the peak position of the distribution is higher than that between the pores. It remained constant even if the distance was increased. From the above results, it is clear that when forming at least two or more through holes 3, it is preferable to set the distance between the through holes 3 to 200 nm or more.

[Example 5 of sample identification method]
Next, a sample identification experiment was performed using three types of polystyrene particles having diameters of 510 nm, 780 nm, and 900 nm as samples, and a device in which seven through holes 3 having a diameter of 1.2 μm were arranged. FIG. 9A is a graph showing the distribution of the peak values of the ion current when the distances of the through holes 3 are all 10 μm, and FIG. 9B is a graph showing the distribution of the peak values of the ion current when the distances of the through holes 3 are all 100 nm. Is. As is clear from the graph of FIG. 9B, when the distance between the through holes 3 was 100 nm, the peaks of the ion currents of the sample sizes of 780 nm and 900 nm overlapped, and it was difficult to identify the sample. On the other hand, as is clear from the graph of FIG. 9A, when the distance of the through hole 3 is increased, the peak distribution of the ion current according to the size of the sample is obtained, and even if the sample has a different size, the identification is performed. We were able to.

[Example 6 of sample identification method]
Next, using 780 nm polystyrene particles as a sample, the ion current was measured using a device in which two through holes 3 having a diameter of 1.2 μm were arranged. 10A shows a distance of the through hole 3 of 100 nm, FIG. 10B shows a distance of the through hole 3 of 1 μm, and FIG. 10C shows a distance of the through hole 3 of 10 μm. The upper part of FIGS. 10A to 10C shows the base current when the ion current is measured. The lower graph is a graph showing the change in the peak value of the ionic current when each sample passes through the through hole 3, as a dot. The part indicated by the arrow in the upper part represents the part (time) at which the base current changed significantly due to the sample being clogged in the through hole 3. The arrow in the lower row also indicates the location (time) at which the sample is clogged in the through hole 3. As shown in FIG. 10A, when the distance between the through holes 3 was short, a remarkable change was found in the peak value of the ion current to be detected after the through holes 3 were clogged. On the other hand, as is clear from FIGS. 10B and 10C, the longer the distance of the through hole 3, the smaller the change in the peak value of the ion current after clogging. From the above results, it is clear that as the distance of the through holes 3 is increased, even if any of the through holes 3 is clogged by the sample, the remarkable effect that the measurement result is less affected is exhibited. became.

In Examples 1 to 6 of the sample identification method, the distance between the through

holes

3 and 10 was up to 10 μm. However, from the measurement results, the method in which the distance between the through

holes

3 and 3 is increased However, it is clear that it is desirable in terms of identification accuracy. Therefore, if necessary, the distance between the through holes 3 may be longer than 10 μm, for example, 11 μm or more, 12 μm or more, 13 μm or more, 14 μm or more.

By implementing the sample identification method using the device disclosed in this specification, the identification accuracy of the sample is improved, and even samples of different sizes can be identified. Therefore, it is useful for the development of analytical devices in the analytical instrument industry.

1, 1a, 1b... Sample identifying device, 2... Substrate, 3... Through hole, 5... First chamber, 6... Second chamber, 7... Ammeter, 8... Analysis unit, 9... Display unit, 10... Program Memory, 11... Control part, 21... First surface, 22... Second surface, 31... First opening, 32... Second opening, 51... First chamber member, 52... First electrode, 53... Lead, 54... Power source, 55... Ground, 61... Second chamber member, 62... Second electrode, 63... Lead, 64... Ground, 100... Identification device, S1, S2... Sample

Claims

A method for identifying a sample in a sample liquid, comprising:
The identification method is
A sample passing step of passing the sample through a through hole formed in the substrate;
An ion current measuring step of measuring a change in ion current when the sample passes through the through hole,
From the value of the measured ion current, a sample identification step of identifying the sample in the sample liquid,
Including at least
Two or more through holes are formed in the substrate, and the through holes are arranged such that the distance between adjacent through holes is at least 200 nm or more.
Sample identification method.
The distance between the adjacent through holes is 1 μm or more,
The sample identification method according to claim 1.
The distance between the adjacent through holes is longer than 10 μ,
The sample identification method according to claim 2.
The sample liquid contains samples of different sizes,
The sample identification method according to any one of claims 1 to 3.
A device used for identifying a sample in a sample liquid,
The device is
Board,
Two or more through holes formed in the substrate,
Including,
The two or more through holes are arranged such that the distance between adjacent through holes is at least 200 nm or more.
Device for sample identification.
The distance between the adjacent through holes is 1 μm or more,
The device for sample identification according to claim 5.
The distance between the adjacent through holes is longer than 10 μm,
The device for sample identification according to claim 6.
A first chamber member forming a first chamber filled with an electrolytic solution at least on the first surface side of the substrate including a through hole;
A second chamber member that forms a second chamber filled with an electrolytic solution by at least the surface including the through hole on the second surface side of the substrate;
The sample identification device according to claim 5, further comprising:
The sample identification device according to claim 8,
A first electrode formed in the first chamber;
A second electrode formed in the second chamber;
An ammeter for measuring an ion current when a sample passes through the through hole,
including,
Sample identification device.