JP7440375B2 - Hole forming method and hole forming device - Google Patents

Description

The present disclosure relates to a hole forming method and a hole forming apparatus.

Techniques using nanopores are being considered as a means of detecting molecules and particles present in aqueous solutions. A nanopore device has a membrane with pores (nanopores) that are about the same size as the molecules or particles to be detected, the upper and lower chambers of the membrane are filled with an aqueous solution, and electrodes are provided in both chambers to make contact with the aqueous solution. It is. During measurement, a detection target to be measured is introduced into one side of the chamber, and a potential difference is applied between the electrodes to cause electrophoresis of the detection target, thereby causing it to pass through the nanopore. By measuring the time change of the ionic current (blocking signal) flowing between the two electrodes at this time, it is possible to detect the passage of the object to be detected and to analyze the structural characteristics of the object to be detected.

In the production of nanopore devices, a method of forming nanopores by processing a semiconductor substrate or semiconductor material using a semiconductor process is attracting attention because of its high mechanical strength. As a method for forming such nanopores, for example, in Non-Patent Document 1, a silicon nitride film (SiNx film) is used as a membrane, and a TEM (transmission electron microscope) device is used to narrow down the irradiation area of an electron beam onto the membrane. discloses that nanopores with a diameter of 10 nm or less can be formed by controlling energy and current.

Furthermore, Patent Document 1 and Non-Patent Documents 2 to 4 disclose nanopore forming methods that utilize the dielectric breakdown phenomenon of membranes. In these methods, first, upper and lower chambers sandwiching a SiNx membrane with no holes are filled with an aqueous solution, electrodes are immersed in the aqueous solution in each chamber, and a high voltage is continuously applied between the two electrodes. When the current between the electrodes suddenly increases (dielectric breakdown of the membrane) and reaches a predetermined cut-off current, it is determined that a nanopore has been formed, and the application of high voltage is stopped to form a nanopore. . This nanopore formation method has the advantage that manufacturing costs are significantly reduced and throughput is improved compared to nanopore formation using a TEM device. Further, in this nanopore formation method, after forming nanopores in the membrane, it is possible to proceed to measurement of the detection target without removing the membrane from the chamber. Therefore, the nanopores are not exposed to pollutants in the atmosphere, which has the advantage of reducing noise during measurement.

One application of measurements using nanopores is the decoding of DNA base sequences (DNA sequencing). That is, this method determines the sequence of four types of bases in a DNA chain by detecting changes in the ionic current passing through the nanopore when DNA passes through the nanopore.

Another application of measurements using nanopores is the detection and counting of specific objects in aqueous solutions. For example, Non-Patent Document 5 describes that PNA and PEG are bound only to DNA of a specific sequence present in an aqueous solution, and changes in ionic current are measured when the DNA modified with PNA and PEG passes through a nanopore. discloses a technique for detecting and counting DNA having a specific sequence.

In such nanopore measurements, the relationship between the size of the object to be detected and the size of the nanopore is important. By using a nanopore that is as close to the size of the object to be detected as possible, the S/N ratio (signal/noise ratio) of the signal (change in ionic current) generated when the object to be detected passes through the nanopore is improved. Since the size of the object to be detected varies, in order to perform highly accurate measurement, it is necessary to accurately form nanopores of various sizes depending on the size of the object to be detected.

International Publication No. 2013/167955

Jacob K Rosenstein, et al., Nature Methods, Vol.9, No.5, 487-492 (2012) Harold Kwok, et al., PloS ONE, Vol.9, No.3, e92880. (2013) Kyle Briggs, et al., Nanotechnology, Vol.26, 084004 (2015) Kyle Briggs, et al., Small, 10(10):2077-86 (2014) Trevor J. Morin, et al., PLoS ONE 11(5):e0154426. doi:10.1371/journal.pone.0154426 Itaru Yanagi, et al., Scientific Reports, 4, 5000 (2014) Christopher E. Arcadia, et al., ACS NANO, 11, 4907-4915 (2017)

When nanopores are formed in a membrane using a dielectric breakdown phenomenon, the sizes of the formed nanopores vary and often deviate from the desired nanopore size. Therefore, when measuring a certain detection target, the probability (yield) of forming nanopores close to the size of the detection target is low.

Therefore, the present disclosure provides a technique for forming pores of a desired size in a film with high yield.

In order to solve the above problems, the pore forming method of the present disclosure is a method of forming pores in a membrane, and includes a first electrode and a second electrode placed across the membrane provided in an electrolytic solution. applying a first current between the electrodes; and applying a first voltage between the first electrode and the second electrode; measuring a flowing second current; and determining whether the second current is greater than or equal to a predetermined threshold; the first current is greater than the threshold; When the second current is smaller than the threshold value, the application of the first current, the application of the first voltage, and the measurement of the second current are repeated.

Further features related to the present disclosure will become apparent from the description herein and the accompanying drawings. Aspects of the present disclosure may also be realized and realized by means of the elements and combinations of various elements and aspects of the following detailed description and appended claims.
The descriptions in this specification are merely typical examples, and do not limit the scope of claims or application examples of the present disclosure in any way.

According to the technology of the present disclosure, holes of a desired size can be formed in a film with a high yield.
Problems, configurations, and effects other than those described above will be made clear by the following description of the embodiments.

1 is a schematic diagram showing a nanopore forming apparatus according to Conventional Example 1. FIG. FIG. 2 is a schematic diagram showing a nanopore forming device having another configuration of Conventional Example 1. FIG. 2 is a diagram for explaining a nanopore forming method according to Conventional Example 1. FIG. 7 is a diagram for explaining a nanopore forming method according to Conventional Example 2. 3 is a schematic diagram showing a nanopore forming apparatus according to Conventional Example 3. FIG. FIG. 7 is a diagram for explaining a nanopore forming method according to Conventional Example 3. FIG. 7 is a diagram for explaining the problems of conventional example 3. FIG. 7 is a diagram for explaining a modification of conventional example 3. 3 is a flowchart of a nanopore forming method according to the first embodiment. FIG. 1 is a schematic diagram showing a nanopore forming device according to a first embodiment. 3 is a time chart for carrying out the nanopore formation method according to the first embodiment. 5 is a time chart for executing a nanopore forming method according to a modification of the first embodiment. 5 is a time chart for executing a nanopore forming method according to a modification of the first embodiment. 7 is a time chart for carrying out the nanopore formation method according to the second embodiment. It is a time chart for carrying out the nanopore formation method based on the modification of 2nd Embodiment. It is a flow chart of a nanopore formation method concerning a 3rd embodiment. 7 is a time chart for carrying out a nanopore forming method according to a third embodiment. 7 is a time chart for executing a nanopore forming method according to a modification of the third embodiment. 7 is a time chart for executing a nanopore forming method according to a modification of the third embodiment. It is a flow chart of a nanopore formation method concerning a 4th embodiment. It is a schematic diagram showing a nanopore formation device concerning a 4th embodiment. It is a time chart for carrying out the nanopore formation method concerning a 4th embodiment. It is a time chart for carrying out the nanopore formation method concerning the modification of a 4th embodiment. It is a time chart for carrying out the nanopore formation method concerning the modification of a 4th embodiment. It is a flow chart of a nanopore formation method concerning a 5th embodiment. 7 is a time chart for carrying out a nanopore forming method according to a fifth embodiment. It is a time chart for carrying out the nanopore formation method concerning the modification of a 5th embodiment. It is a time chart for carrying out the nanopore formation method concerning the modification of a 5th embodiment. FIG. 7 is a diagram showing an example of the shape of a pulse current according to a sixth embodiment. It is a schematic diagram showing a nanopore formation device concerning a 7th embodiment.

Embodiments of the present disclosure will be described in detail below with reference to the drawings. In all the figures of the present disclosure, parts having the same functions are given the same reference numerals, and repeated explanations thereof are omitted as much as possible. The structure and materials of the device described in the embodiments are examples for embodying the idea of the present disclosure, and the materials, dimensions, etc. are not strictly specified. Further, the specific voltage values, current values, and application times of current and voltage described in the embodiments are examples for embodying the idea of the present disclosure, and are not strictly specified.

In the present disclosure, the "pore forming method" includes a method of creating pores in a membrane with no pores, and a method of widening pores already formed in the membrane to a desired size. or a method of adjusting.

[Conventional example 1]
First, conventional methods for forming nanopores using dielectric breakdown (Conventional Examples 1 to 3) will be described.

FIG. 1 is a schematic diagram showing a nanopore forming apparatus 1000 for realizing a nanopore forming method according to Conventional Example 1. Nanopore forming device 1000 includes membrane 101, electrodes 104 and 105, chambers 110 and 111, and control circuit 2000.

Chambers 110 and 111 are separated by membrane 101. As the membrane 101, for example, a silicon nitride film (SiN film) with a thickness of 3 to 20 nm can be used. The chamber 110 houses an aqueous solution 102, and the chamber 111 houses an aqueous solution 103. As the aqueous solutions 102 and 103, for example, a KCl aqueous solution can be used. The chamber 110 is provided with a solution inlet 106 and a solution outlet 107, and the chamber 111 is provided with a solution inlet 108 and a solution outlet 109. An electrode 104 is in contact with the aqueous solution 102 in the chamber 110, and an electrode 105 is in contact with the aqueous solution 103 in the chamber 111. Electrodes 104 and 105 are connected to control circuit 2000. As the electrodes 104 and 105, for example, Ag/AgCl electrodes can be used.

Control circuit 2000 includes a voltage source 2001 and an ammeter 2002. Control circuit 2000 drives voltage source 2001 to apply an arbitrary voltage between electrodes 104 and 105. Further, the control circuit 2000 measures the current flowing between the electrodes 104 and 105 using the ammeter 2002, and records the measured current value in a storage device (not shown). Further, the control circuit 2000 can change the voltage applied between the electrodes 104 and 105 based on information on the measured current value.

FIG. 2 is a schematic diagram showing a nanopore forming device 1001 having another configuration. In the nanopore forming device 1001, the membrane 101 is supported by a support substrate 112. Further, a sealing material 113 is disposed between the membrane 101 and the chamber 110, and a sealing material 114 is disposed between the support substrate 112 and the chamber 111. The other configurations are the same as the nanopore forming apparatus 1000 in FIG. As the material of the support substrate 112, silicon (Si) can be used, for example. The sealing materials 113 and 114 are, for example, O-rings, and prevent leakage of the aqueous solution in the chambers 110 and 111, respectively.

Generally, a nanopore forming device has a support substrate 112 and sealants 113 and 114, like the nanopore forming device 1001 in FIG. Hereinafter, in this specification, a simplified diagram like FIG. 1 in which the support substrate 112 and the sealants 113 and 114 are omitted will be used for the sake of simplification of illustration.

FIG. 3 is a diagram for explaining a nanopore forming method according to Conventional Example 1. The nanopore formation method according to Conventional Example 1 is referred to as a "constant voltage application method." Note that this method is also disclosed in Non-Patent Document 2. The constant voltage application method is such that when voltage V ₁ (interelectrode voltage) is applied between electrodes 104 and 105, the current flowing between electrodes 104 and 105 (interelectrode current I) becomes approximately I _th (threshold current). This is a method to form nanopores of a large size.

As shown in FIG. 3, in the constant voltage application method, a control circuit 2000 is used to apply a constant voltage V ₁ between electrodes 104 and 105 (upper diagram), and at that time, the current flowing between electrodes 104 and 105 is controlled. Measure (see below). Then, the control circuit 2000 stops applying the voltage between the electrodes 104 and 105 when the measured interelectrode current I becomes equal to or greater than I _th . The voltage V ₁ is high enough to cause dielectric breakdown of the membrane 101 .

Since it is not possible to measure the interelectrode current I completely continuously, the interelectrode current I is recorded at every sampling interval ts. The recorded interelectrode current I is shown in the lower part of FIG. 3 as a black dot. As shown in the lower diagram of FIG. 3, the measured interelectrode current I rapidly increases after a certain time. At this point, the membrane 101 undergoes dielectric breakdown and fine pores are generated in the membrane 101. Thereafter, the voltage V ₁ continues to be applied to the membrane 101, and when the interelectrode current I reaches I _th or more, the application of the voltage V ₁ is stopped. Thereby, it is possible to form a nanopore through which a current having a size of about I _th flows when a voltage V ₁ is applied.

As mentioned earlier, the interelectrode current I cannot be measured completely continuously, but is measured at a certain sampling interval ts, so the interelectrode current I and I _th at a certain sampling point are completely It is unlikely that they will match, and in reality, the voltage application will be stopped when the measured (recorded) interelectrode current I exceeds I _th . Furthermore, it takes a certain amount of time for the control circuit 2000 to determine whether the measured interelectrode current I exceeds I _th . When the control circuit 2000 determines that the measured interelectrode current I exceeds I _th , it takes a certain amount of time to stop applying the voltage between the electrodes 104 and 105.

That is, in the constant voltage application method, even after the interelectrode current I reaches I _th , the voltage V ₁ continues to be applied to the membrane 101 for a certain period of time, and the pores continue to expand during that time. The pore expands due to Joule heat (current passing through the pore x applied voltage). The larger the pore, the larger the current flowing when voltage _V1 is applied, and the larger the Joule heat. As the Joule heat increases, the speed at which the pore size expands becomes even faster. In this manner, in the constant voltage application method, even after the interelectrode current I reaches I _th , the voltage V ₁ continues to be applied to the membrane 101 for a certain period of time, during which time the pores expand at an accelerated rate. Therefore, the pore that is actually formed is larger than the pore that is large enough to allow current I _th to flow when voltage V ₁ is applied.

As described above, in the constant voltage application method, high Joule heat continues to be generated even after the pores reach a desired size. The degree to which the pores expand due to this Joule heat differs from membrane to membrane (even if membranes of the same material and thickness are used). Sometimes the pores expand all at once, and sometimes they don't. Therefore, variations may occur in the size of the pores formed.

Further, it is difficult to accurately calculate the size of the nanopore from the interelectrode current I when a high voltage (V ₁ ) high enough to cause dielectric breakdown of the membrane 101 is applied. The reason for this is as follows. The interelectrode current I after the nanopore is formed can be expressed as I=I _np +I _noise . I _np is the current passing through the nanopore (nanopore current). If the ion concentration and ion mobility of the aqueous solutions 102 and 103, the thickness and material of the membrane 101, the voltage applied to the electrodes 104 and 105, and the nanopore current I _np are known, the size of the pore can be estimated. can. On the other hand, I _noise is a current that causes noise (noise current) other than the nanopore current I _np . It may be an electric current resulting from a reaction or an exchange of charges. The noise current I _noise increases as the applied voltage increases. Further, the noise current I _noise differs depending on the membrane 101 used (even if membranes of the same material and thickness are used). Further, the noise current I _noise changes with the time of voltage application. In the constant voltage application method, a high voltage (V ₁ ) high enough to cause dielectric breakdown of the membrane 101 is applied, so the current flowing between the electrodes 104 and 105 includes components of the noise current I _noise other than the nanopore current I _np . big. Therefore, since it is not possible to measure only the nanopore current I _np , it is difficult to estimate the exact size of the pore from the interelectrode current I. Furthermore, since the noise current I _noise differs for each membrane 101, even if pores through which current I _th flows when voltage V ₁ is applied to each of multiple membranes 101 can be formed, the size of these pores will vary. Variations may occur.

[Conventional example 2]
FIG. 4 is a diagram for explaining a nanopore forming method according to Conventional Example 2. The nanopore forming method according to Conventional Example 2 can be realized using an apparatus similar to the nanopore forming apparatus 1000 of Conventional Example 1. The nanopore formation method according to Conventional Example 2 is referred to as a "pulse voltage application method." Note that this method is also disclosed in Non-Patent Document 6. In the pulse voltage application method, when a voltage V ₂ is applied between the electrodes 104 and 105, the current flowing between the electrodes 104 and 105 (interelectrode current I) is approximately I _th ' (threshold current). This is a method for forming nanopores.

As shown in FIG. 4, in the pulse voltage application method, the control circuit 2000 is used to repeatedly apply a pulse voltage of high voltage _V1 and measure current when applying low voltage _V2 . The control circuit 2000 stops applying the pulse voltage of the high voltage V ₁ when the interelectrode current I measured when the low voltage V ₂ is applied becomes equal to or higher than I _th ′.

The pulse voltage application method is different from the constant voltage application method in that a pore is formed such that the interelectrode current I becomes approximately I _th ' when a low voltage V ₂ is applied. Specifically, the value of the low voltage _V2 is set such that the noise current _Inoise is sufficiently smaller than the nanopore current _Inp among the interelectrode current I= _Inp + _Inoise . Therefore, after the nanopores are formed in the membrane 101, the size of the nanopores can be almost accurately calculated from the interelectrode current I (≈I _np ) when the low voltage V ₂ is applied. Therefore, the variation in pore size caused by the variation in the noise current I _noise , which may occur in the constant voltage application method of Conventional Example 1, is resolved.

However, the pore that is actually formed may be larger than the pore that allows a current of magnitude I _th ' to flow when the low voltage V ₂ is applied. This means that at some point during the application of the high voltage pulse voltage _V1 (in the example of FIG. 4, at some point during the application of the fourth pulse voltage), the size of the nanopore changes to the lower voltage. When V ₂ is applied, the interelectrode current I reaches a magnitude of I _th ', but even after that, the high voltage V ₁ is applied between the electrodes 104 and 105 until the application of the pulse voltage is finished. This is so that it continues to be used. During that time, the pore continues to expand due to Joule heat (current passing through the pore x applied voltage). The larger the pore, the larger the current flowing when the high voltage _V1 is applied, and the larger the Joule heat. As the Joule heat increases, the pore expands even faster. In this way, in the pulsed voltage application method, even after the pores reach a desired size, the high voltage _V1 continues to be applied to the membrane 101 until the end of the pulse, during which time the pores expand at an accelerated rate.

As described above, in the pulse voltage application method, high Joule heat continues to be generated even after the pores reach a desired size. The degree to which the pores expand due to this high Joule heat differs from membrane to membrane each time (even when using the same material and membrane thickness). Sometimes the pores expand all at once, and sometimes they don't. Therefore, variations may occur in the size of the pores formed.

In the case of the pulse voltage application method, fine pores are generated in the membrane 101 at some point during the application of a certain pulse (in the example of FIG. 4, at some point during the application of the third pulse voltage). After that, the interelectrode current I when the low voltage V ₂ is applied becomes clearly larger than the previous measurement value (fourth plot from the left). Here, after the fine pores are generated in the membrane 101, the high voltage _V1 continues to be applied until the pulse ends, and during that time, the generated pores are heated by Joule heat (current passing through the pores x applied voltage). Continue to expand. The larger the pore, the larger the current flowing when the high voltage _V1 is applied, and the larger the Joule heat. As the Joule heat increases, the pore expands even faster. In this way, the size of the pores increases at an accelerating rate after the minute pores are generated in the membrane until the pulse voltage that is being applied at that time ends. As a result, at the end of the application of the pulsed voltage that generated the micropores in the membrane, the nanopores are already much larger than the desired size (i.e., nanopores of such a size that current I _th flows when a low voltage V ₂ is applied). There is also a possibility that it becomes a pore.

[Conventional example 3]
FIG. 5 is a schematic diagram showing a nanopore forming apparatus 1002 for realizing the nanopore forming method according to Conventional Example 3. The nanopore formation method according to Conventional Example 3 is referred to as a "constant current application method." Note that this method is also disclosed in Non-Patent Document 7.

Nanopore forming device 1002 according to Conventional Example 3 is almost the same as nanopore forming device 1000 of Conventional Examples 1 and 2, but the function of control circuit 3000 is different from control circuit 2000.

Control circuit 3000 includes a current source 3001 and a voltmeter 3002. The control circuit 3000 applies (flows) an arbitrary current between the electrodes 104 and 105 by driving the current source 3001. Further, the control circuit 3000 measures the voltage between the electrodes 104 and 105 using the voltmeter 3002, and records the measured voltage value in a storage device (not shown). Further, the control circuit 3000 can change the applied current based on information on the measured voltage value.

FIG. 6 is a diagram for explaining a nanopore forming method according to Conventional Example 3. The constant current application method is a method of forming a nanopore of such a size that the voltage between the electrodes becomes approximately V _th (threshold voltage) when a current I ₁ is applied between the electrodes 104 and 105.

As shown in FIG. 6, in the constant current application method, a control circuit 3000 is used to apply a current so that a constant current _I1 flows between electrodes 104 and 105, and after pores are formed in membrane 101, When the inter-electrode voltage becomes equal to or less than a predetermined voltage V _th , current application is stopped.

A detailed explanation of the phenomenon of the constant current application method is as follows. That is, until a pore is formed, the membrane 101 is charged with electric charge by flowing a constant current _I1 between the electrodes 104 and 105, and as a result, the voltage between the electrodes increases according to the relational expression Q=CV. To go. In other words, membrane 101 serves as a capacitor. Then, after the pores are formed at a certain point, current begins to flow through the pores, so the inter-electrode voltage required to flow a constant current _I1 drops rapidly. Thereafter, as the formed pores expand, the voltage between the electrodes decreases, and when the voltage between the electrodes becomes V _th or less, the application of current between the electrodes is stopped.

In the constant current application method, the voltage between the electrodes is automatically lowered immediately after the pores are formed. Therefore, since the current _I1 applied between the electrodes is constant, Joule heat (current passing through the pore x applied voltage) decreases immediately after the pore is formed. And the more the pores expand, the lower the Joule heat. Therefore, rapid expansion of the pore is unlikely to occur after it is formed. On the other hand, in the constant voltage application method and the pulsed voltage application method, Joule heat continues to increase during the pore expansion process, so the pore expansion speed is fast. Therefore, in the constant current application method, compared to the constant voltage application method and the pulsed voltage application method, it is possible to create a pore of a desired size (such that the voltage between the electrodes becomes V _th when the current applied between the electrodes is I ₁ ). The pores can be formed closer to each other. Furthermore, in the constant current application method, rapid expansion of the pores is unlikely to occur, so the pores do not suddenly become larger than a desired size. Therefore, variations in the size of the pores formed are reduced.

However, the present inventors discovered a problem with the constant current application method. FIG. 7 is a diagram for explaining the problems of the constant current application method. A constant current _I1 is applied between the electrodes 104 and 105, and after pores are generated in the membrane 101, the application of the current _I1 between the electrodes is stopped when the interelectrode voltage becomes _Vth or less, thereby obtaining the desired magnitude. Assume that a large pore (a pore of such a size that the voltage between the electrodes becomes V _th when the current applied between the electrodes is I ₁ ) is formed. In this case, as shown in FIG. 7, it was found that the inter-electrode voltage may not reach V _th even after a long period of time, or it may take a long time for the inter-electrode voltage to reach V _th . The likelihood of this phenomenon occurring varies depending on how V _th and I ₁ are set and the material and thickness of the membrane 101, but it has been found that it is more likely to occur when V _th is set low. If the interelectrode voltage does not reach V _th even after a long period of time, it means that the pores do not expand to the desired size. The reason for this is that, as mentioned above, in the constant current application method, Joule heat decreases after pores are generated, and the more the pores expand, the more Joule heat decreases. Therefore, once the pores expand to a certain extent, it becomes impossible to obtain enough Joule heat to expand the pores. Then, pore expansion almost stops.

FIG. 8 is a diagram for explaining a modification of the constant current application method. As a solution to the problem of the constant current application method described above, a method can be considered in which the current applied between the electrodes and the threshold value of the interelectrode voltage are made sufficiently large, as shown in FIG. In other words, when forming a pore of such a size that the interelectrode voltage is V _th when the interelectrode current is I ₁ , for example, the interelectrode current is 10 × I ₁ and the interelectrode voltage threshold is 10 × V Set as _th . As a result, the Joule heat after the pore is formed is larger than the Joule heat after the pore is formed when the current applied between the electrodes is set to _I1 and the threshold value of the interelectrode voltage is set to _Vth . . Note that the magnitude of the voltage between the electrodes when the pores are formed does not depend much on the magnitude of the constant current applied between the electrodes, so the pores tend to expand. Furthermore, by increasing the threshold value of the interelectrode voltage, the number of cases in which the interelectrode voltage does not reach the threshold value even after a long period of time is reduced.

However, in this modified example of the constant current application method, variations may occur in the sizes of the resulting pores. As mentioned above, the interelectrode current can be expressed as I=I _np +I _noise . When the voltage between the electrodes is small, the noise current I _noise is sufficiently smaller than the nanopore current I _np . However, as the voltage between the electrodes increases, the value of the noise current I _noise increases. Therefore, a pore of such size that the inter-electrode voltage is V _th when the inter-electrode current is I ₁ , and a pore of such size that the inter-electrode voltage is 10 × V _th when the inter-electrode current is 10 × I ₁ . The pores differ in size. Therefore, even if a pore is formed by setting the interelectrode current to 10 × I ₁ and the interelectrode voltage threshold to 10 × V _th , the pore of the desired size (when the interelectrode current is I ₁ A different pore size is formed such that the voltage is V _th . In addition, since the noise current I _noise changes from time to time for each membrane 101, when pores are formed by increasing the threshold value (V _th ) of the interelectrode current and the interelectrode voltage, there is a large variation in the size of the resulting pores. Become.

[First embodiment]
<Nanopore formation method>
FIG. 9 is a flowchart of the nanopore formation method according to the first embodiment. The nanopore forming method according to the first embodiment is a method of forming a nanopore having a size such that the inter-electrode voltage is about V _th when the inter-electrode current is I ₁ . In the following, each step will be explained assuming that the operator is the main person responsible for the operation, but each step can also be executed by the control computer driving each element of the control circuit.

(Step S1)
The operator drives the current source and applies (flows) a pulse current I ₂ that is larger than the current I ₁ and has a pulse width (time width) of t _n between the electrodes provided with the membrane in between. The pulse width t _n can be set, for example, to 1 μs or more and 10 s or less, or 1 ms or more and 1 s or less.

(Step S2)
The operator drives the voltage source, applies a voltage V _th between the electrodes provided across the membrane, and measures the current I between the electrodes using an ammeter. The voltage V _th can be set to a value such that the noise current I _noise is sufficiently smaller than the nanopore current I _np . Thereby, the size of the nanopore can be almost accurately calculated from the interelectrode current I (≈I _np ) when the voltage V _th is applied. Therefore, variations in pore size caused by variations in the noise current I _noise can be reduced.

Specifically, the voltage V _th can be set in a range of 1 mV or more and 1V or less. Alternatively, the voltage V _th can be set to be 1 mV or more and so that the electric field applied to the membrane when the voltage V _th is applied between the electrodes is 0.3 V/nm or less. Alternatively, the voltage V _th can be set to a voltage such that when a voltage is applied to the membrane in a state where no pores are formed, the current flowing through the membrane becomes 1 nA or less. Alternatively, the voltage V _th can be set to a voltage such that when a voltage is applied to the membrane in a state where no pores are formed, the current flowing through the membrane is 1/5 or less of I ₁ .

(Step S3)
The operator determines whether the measured interelectrode current I is greater than or equal to the current I ₁ (threshold). If the interelectrode current I is smaller than the current _I1 (No), the process moves to step S4. If the interelectrode current I becomes equal to or greater than the current _I1 (Yes), the operator determines that a pore of a desired size has been formed, and ends the process.

(Step S4)
The operator sets the number of times of application of the pulse current _I2 to n=n+1 and returns to step S1. In this embodiment, when the pulse width of the n-th pulse current I ₂ is t _n and the pulse width of the n+1-th pulse current I ₂ is t _n+1 , t _n =t _n+1 .

As described above, the nanopore formation method according to the first embodiment uses a pulsed current I ₂ (first current) between electrodes placed across a membrane provided in an aqueous solution (electrolyte). applying a voltage V _th (first voltage) between the electrodes and measuring the inter-electrode current I ( _second current); The pulse current _I2 is set larger than the current I1, and when the interelectrode current I is smaller than the current _I1 , the pulse current _I2 is set to be larger than the current _I1 . The application of the voltage V _th and the measurement of the interelectrode current I are repeated. In addition, when the interelectrode current I becomes the current I ₁ or more, a pore of a desired size (a pore of a size such that the interelectrode voltage becomes approximately V _th when the interelectrode current is I ₁ (threshold value)) It can be determined that pores) have been formed. Thereby, pores of a desired size can be formed with a high yield. In this specification, the method of forming nanopores according to this embodiment is sometimes referred to as a "pulse current application method."

<Nanopore formation device>
FIG. 10 is a schematic diagram showing a nanopore forming apparatus 100 for realizing the nanopore forming method according to the first embodiment. The nanopore forming device 100 includes a membrane 101, electrodes 104 and 105 (first electrode and second electrode), chambers 110 and 111 (first liquid tank and second liquid tank), and a control circuit 200.

Chambers 110 and 111 are separated by membrane 101. As the membrane 101, for example, a silicon nitride film (SiN film) with a thickness of 3 to 20 nm can be used. The chamber 110 houses an aqueous solution 102, and the chamber 111 houses an aqueous solution 103. As the aqueous solutions 102 and 103, for example, a KCl aqueous solution can be used. The chamber 110 is provided with a solution inlet 106 and a solution outlet 107, and the chamber 111 is provided with a solution inlet 108 and a solution outlet 109. An electrode 104 is in contact with the aqueous solution 102 in the chamber 110, and an electrode 105 is in contact with the aqueous solution 103 in the chamber 111. Electrodes 104 and 105 are connected to control circuit 200. As the electrodes 104 and 105, for example, Ag/AgCl electrodes can be used.

The control circuit 200 includes a current source 201, a variable voltage 202, an ammeter 203, and a switch 204. By connecting switch 204 to contact A, electrodes 104 and 105 are connected to a circuit with current source 201 (first circuit). By connecting switch 204 to contact B, electrodes 104 and 105 are connected to a circuit (second circuit) with variable voltage 202 and ammeter 203.

FIG. 11 is a time chart for executing the nanopore forming method according to the first embodiment using the nanopore forming apparatus 100. As shown in FIG. 11, first, after connecting the switch 204 to the contact A, the current source 201 is driven to apply a pulse current of _I2 , which is larger than the current _I1 , between the electrodes 104 and 105. The pulse width of the pulse current is t _n . The pulsed current is constant in magnitude _I2 and is a square wave while being applied between electrodes 104 and 105. In this specification, such a pulsed current is referred to as a "constant current pulse."

After the application of the pulse current is finished, the switch 204 is switched from contact A to contact B. Then, by driving the variable voltage 202, a voltage V _th is applied between the electrodes 104 and 105, and the current I between the electrodes at that time is measured by the ammeter 203. If the interelectrode current I is not greater _than the current I1, the switch 204 is switched from the contact B to the contact A, and then a pulse current _I2 larger than the current _I1 is applied between the electrodes 104 and 105. The pulse width at that time is t _n+1 , and t _n =t _n+1 . The above procedure is repeated until the interelectrode current I when the voltage V _th is applied between the electrodes 104 and 105 becomes the current I ₁ or more, and the interelectrode current when the voltage V _th is applied between the electrodes 104 and 105 is The process ends when I becomes I1 or _more .

In the example shown in FIG. 11, the timing at which the pulse current rises is after a certain period of time has elapsed after switching the switch 204 from contact B to contact A. The timing of switching to contact A may be simultaneous. Similarly, in the example shown in FIG. 11, the timing for switching the switch 204 from contact A to contact B is after a certain period of time has passed after the pulse current falls, but the switch 204 is switched from contact A to contact B. The switching timing and the falling timing of the pulse current may be the same.

After switching the switch 204 from contact A to contact B, the voltage between electrodes 104 and 105 is temporarily set to 0V, thereby eliminating the potential difference generated between electrodes 104 and 105 due to the application of the pulse current. Thereby, when applying the voltage V _th between the electrodes 104 and 105 and measuring the inter-electrode current I at that time, the inter-electrode current I can be measured more accurately.

In the constant current application method (Conventional Example 3), when forming a nanopore of a size such that the interelectrode voltage is approximately V _th when the interelectrode current is I ₁ , a constant current of magnitude I ₁ is applied between the electrodes. was applied. On the other hand, in the pulsed current application method, when forming a nanopore of a size such that the interelectrode voltage is approximately V _th when the interelectrode current is I ₁ , a constant current pulse of I ₂ larger than the current I ₁ is applied. is being applied. Therefore, the Joule heat of the current passing through the pore is I ₂ ×V = I ₂ ×I ₂ ×R (R is the resistance of the nanopore), and the Joule heat of the constant current application method is I ₁ ×V = I ₁ ×I ₁ ×R (R is the resistance of the nanopore). Therefore, in the pulsed current application method, the pores tend to expand, and it is possible to reduce the possibility that the pores will not expand to the desired size after pore formation, or that it will take a long time to reach the desired pore size.

As a result of intensive study by the present inventors, by setting the pulse current I ₂ to satisfy I ₁ ×1.5≦I ₂ , there is a possibility that the pores will not expand to the desired size after pore formation, or that the desired size will be reduced. It has been found that the possibility of it taking a very long time to reach the pore size can be greatly reduced. Furthermore, it has been found that these possibilities can be further reduced by setting the pulse current I ₂ to satisfy I ₁ ×2≦I ₂ . Furthermore, these possibilities can be further reduced by satisfying 0.3V/nm < pulse current I ₂ × pulse width t _n ÷ interelectrode capacitance (C) ÷ membrane thickness (nm) < 2V/nm. Do you get it.

Furthermore, in this embodiment, since a constant current pulse is used, the inter-electrode voltage continues to decrease immediately after the pore opens during application of a certain pulse current. In the example of FIG. 11, pores are formed during the application of the fifth pulse current, and the interelectrode voltage continues to decrease immediately after the pores are formed. Therefore, the Joule heat continues to decrease from immediately after the pores are formed until the pulse current (fifth pulse current) applied at that time ends. Naturally, also in the subsequent process of enlarging the pores to a desired size by applying a pulse current, the Joule heat decreases as the pores enlarge. Therefore, it is unlikely that the pores will expand rapidly after a certain pulse current is applied, resulting in the formation of pores much larger than the desired size. That is, in the method of this embodiment, rapid expansion of the pores is unlikely to occur after a certain pulse current is applied, so the pores will not grow significantly beyond the desired size. Therefore, variations in the size of the pores formed are reduced.

In contrast, in the pulse voltage application method (Conventional Example 2), even after a pore is formed during the application of a certain pulse voltage, the same voltage continues to be applied until the application of that pulse voltage ends. Furthermore, after the pore is formed, the pore expands until the application of the pulse voltage that is being applied at that time is terminated, thereby increasing the current flowing through the pore. As a result, after the pore is formed, Joule heating continues to increase until the pulse voltage is terminated. This also applies to the process of enlarging the formed pore by applying an additional pulse voltage to adjust the pore to a desired size.

<Modification 1 of the first embodiment>
FIG. 12 is a time chart for executing the nanopore forming method according to the first modification of the first embodiment using the nanopore forming apparatus 100. In Modification 1, the set value of the output current of the current source 201 is fixed to _I2 , and by switching the switch 204 between contact A and contact B, a pulse current is effectively applied between the electrodes 104 and 105. be done. When switch 204 is connected to contact B, the circuit connected to current source 201 is not closed. Therefore, even if the set value of the output current of the current source 201 is left at _I2 , no current flows from the current source 201. Instead, the output voltage of current source 201 increases to a limit value. In other words, the period (time width) during which the switch 204 is connected to the contact point A is the time width t _n of the pulse current. When switch 204 is connected to contact B, the voltage is set to V _th and interelectrode current I is measured. If the measured interelectrode current I has not reached the current _I1 , switch 204 is switched from contact B to contact A to flow current between electrodes 104 and 105, and after a certain period of time has passed, switch 204 is switched to contact A. to contact B, and measure the interelectrode current I at voltage V _th . By repeating this, the pulse current application method is executed.

Modification 1 can effectively apply a pulsed current between the electrodes 104 and 105 without making the set value of the output current of the current source 201 pulse-like, so that the operation for the current source 201 is similar to that of the first embodiment. It is simpler than (Fig. 11). On the other hand, in Modification 1, when the switch 204 is switched from the contact B to the contact A, a large voltage close to the limit value of the output voltage of the current source 201 is instantaneously applied. This instantaneous high voltage can cause the pore to become larger than expected. Conversely, in the first embodiment, the pulse current rises after the switch 204 is switched from contact B to contact A, so a large voltage is not instantaneously applied.

<Modification 2 of the first embodiment>
FIG. 13 is a time chart for executing the nanopore forming method according to the second modification of the first embodiment using the nanopore forming apparatus 100. In this modification example 2, after switching the switch 204 from contact B to contact A, a constant current pulse of magnitude I ₂ is applied, and while the constant current pulse of magnitude I ₂ is being applied, the switch 204 is switched from contact A to Switch to contact B. Thereafter, while the switch 204 is connected to contact B, the set value of the output current of the current source 201 is set to 0. Furthermore, while the switch 204 is connected to the contact B, a voltage V _th is applied between the electrodes 104 and 105, and the inter-electrode current I flowing at that time is measured. If the interelectrode current I is smaller than _I1 , switch 204 is switched from contact B to contact A again, and the same procedure is performed. Finally, while the switch 204 is connected to the contact B, a voltage V _th is applied between the electrodes 104 and 105, and the procedure ends when the current I between the electrodes at that time becomes I ₁ or more.

The time width t _n of the pulse current I ₂ in Modification 2 is from when the set value of the output current of the current source 201 reaches I ₂ until the switch 204 switches from contact A to contact B. In other words, if you want to simultaneously stop the pulse current applied between the electrodes 104 and 105 at the same time the switch 204 is switched from contact A to contact B, the timing for switching the switch 204 from contact A to contact B is set by the current source 20 This can be achieved by setting the set value of the output current earlier than the timing at which I falls from ₂ to 0. As a result, the pulse current can be automatically reduced by switching the switch 204 without being aware of the timing of the fall of the pulse current by driving the current source 201, which simplifies the operation.

[Second embodiment]
In the first embodiment, it has been explained that the pulse width (time width) of the pulse current is the same in all times, and the magnitude of the pulse current is also the same in all times. In the second embodiment, in order to shorten the time required to form a pore of a desired size, a method is proposed in which the pulse width is increased as the number of pulse current applications is increased. The nanopore forming method according to the second embodiment can be realized using the nanopore forming apparatus 100 described in the first embodiment.

FIG. 14 is a time chart for executing the nanopore forming method according to the second embodiment using the nanopore forming apparatus 100. As shown in FIG. 14, the pulse width of the n-th pulse current is t _n , the pulse width of the n+1-th pulse current is t _n+1 , and t _n <t _n+1 . Other processes are the same as those in the first embodiment and its modifications, so description thereof will be omitted.

In this way, by increasing the pulse width of the pulse current with the number of times the pulse current is applied, the amount of stress applied to the membrane or pore by one application of the pulse current increases with the number of times the pulse current is applied. Therefore, the number of times a pulse current must be applied until a pore of a desired size is formed is reduced, and as a result, the time required for a pore of a desired size to be formed is shortened.

The method of the second embodiment is effective, for example, for problems such as those described below. The current application time required until pores are formed in the membrane 101 varies even when membranes of the same thickness and material are used. Therefore, pores may be formed by applying the current _I2 for a short time, or may not be formed if the current I2 is not applied for a long time. In particular, if a pore is not formed unless the current _I2 is applied for a long time, the pulse width _tn is short and set to _tn = tn ₊₁ (the pulse width is constant regardless of the number of times the pulsed current is applied). Therefore, it takes a long time for pores to form in the membrane. On the other hand, by setting the pulse width to t _n <t _n+1 as in this embodiment, the time until pore formation can be shortened.

<Modification of the second embodiment>
In the second embodiment, in order to shorten the time required to form a pore of a desired size, the pulse width was increased as the number of pulse current applications was increased. In contrast, in a modification of the present embodiment, a method is proposed in which the time required to form a pore of a desired size is shortened by increasing the magnitude of the pulse current as the number of times of application increases.

FIG. 15 is a time chart for executing the nanopore forming method according to the modified example of the second embodiment using the nanopore forming apparatus 100. As shown in FIG. 15, the magnitude of the n-th pulse current is I _n , the magnitude of the n+1-th pulse current I _n+1 , and I _n <I _n+1 . However, the minimum value of the pulse current is larger than I ₁ (threshold value).

The larger the pulse current, the higher the probability that pores will be formed during the application of the pulse current, so pores can be formed in the membrane 101 with fewer pulse applications. Also, the larger the pulse current, the more likely the pore will expand during the application of that pulse. Therefore, the number of pulses that must be applied until a pore of a desired size is formed is reduced, and as a result, the time required for a pore of a desired size to be formed is shortened.

[Third embodiment]
In the first and second embodiments, a method for creating pores of a desired size in a membrane without pores was described. In the third embodiment, we propose a method for more accurately adjusting the pores created in the membrane to a desired size. In the third embodiment, a pore of a desired size is a pore of a size such that when the interelectrode voltage is V _th , the interelectrode current is approximately I ₁ ′ (second threshold current). .

FIG. 16 is a flowchart of a nanopore forming method according to the third embodiment.

(Steps S11 to S14)
Steps S11 to S14 are almost the same as steps S1 to S4 described in the first embodiment, so their explanation will be omitted. However, the pulse current applied between the electrodes 104 and 105 in step S11 will be referred to as "first pulse current _I2 ." In step S14, if the interelectrode current I measured when the voltage V _th is applied becomes equal to or higher than the current I ₁ (first threshold current) (Yes), the process moves to step S15.

(Step S15)
The operator drives the current source to generate a pulse width that is larger than the current I ₁ ' (second threshold current) and smaller than the first pulse current I ₂ between the electrodes provided across the membrane. A second pulsed current I ₂ ′ of t _m is applied. The pulse width t _m can be set, for example, to 1 μs or more and 10 s or less, or 1 ms or more and 1 s or less.

(Step S16)
The operator drives the voltage source, applies a voltage V _th between the electrodes provided across the membrane, and measures the current I between the electrodes using an ammeter. The voltage V _th at this time can be set similarly to the first embodiment.

(Step S17)
The operator determines whether the measured interelectrode current I is greater than or equal to the current I ₁ '. If the interelectrode current I is smaller than the current I ₁ ' (No), the process moves to step S18. If the interelectrode current I becomes equal to or greater than the current I ₁ ' (Yes), the operator determines that the pore has reached the desired size and ends the process.

(Step S18)
The operator sets the number of applications of the second pulse current I ₂ ' to m=m+1 and returns to step S15. In this embodiment, when the pulse width of the m-th second pulse current I ₂ ′ is t _m , and the pulse width of the m+1-th second pulse current I ₂ ′ is t _m+1 , t _m = t It can be _m+1 .

FIG. 17 is a time chart for executing the nanopore forming method according to the third embodiment using the nanopore forming apparatus 100. In FIG. 17, a time chart of the switch 204 is omitted. As shown in FIG. 17, first, by driving the current source 201, a first pulse current _I2 larger than the current _I1 is applied between the electrodes 104 and 105, and then the switch 204 is switched from contact A to contact B. . Thereafter, a voltage V _th is applied by driving the variable voltage 202, and the interelectrode current I at that time is measured by the ammeter 203. Repeat this process, and when the interelectrode current I when voltage V _th is applied becomes greater than or equal to current I ₁ , change the set value of the output current of current source 201 so that the current I is smaller than I ₂ and larger than current I ₁ ′. 2 pulse current I ₂ ' is applied. Thereafter, the switch 204 is switched from contact A to contact B, voltage V _th is applied by driving the variable voltage 202, and interelectrode current I at that time is measured. The application of the second pulse current I ₂ ′ and the current measurement when applying the voltage V _th are repeated, and the process ends when the interelectrode current I becomes equal to or higher than the current I ₁ ′.

As described above, in this embodiment, the threshold current is provided in two stages. That is, there are two currents: current I ₁ and current I ₁ '. Then, from the time when the interelectrode current I becomes equal to or higher than the current _I1 , the pulse current is decreased from _I2 to _I2 '. This makes it possible to reduce the amount by which the pore expands each time a pulse current is applied. In other words, it is possible to reduce the amount of increase in interelectrode current I when voltage V _th is applied, which increases each time a pulse current is applied. Therefore, it is possible to form a pore having a size closer to a desired size (a pore having a size such that the interelectrode current is about I ₁ ' when the interelectrode voltage is V _th ).

Further, pulse current I ₂ is set to satisfy I ₁ ×1.5≦I ₂ , pulse current I ₂ ′ is set to satisfy I ₁ ′×1.5≦I ₂ , and I ₂ By setting such that >I ₂ ′ and I ₁ ′>I ₁ are satisfied, pores having a size closer to a desired size can be formed.

<Modification 1 of the third embodiment>
In the third embodiment, it was explained that the pulse width and magnitude of the first pulse current are the same in all times, and the pulse width and magnitude of the second pulse current are also the same in all times. . On the other hand, in Modification 1 of the third embodiment, following the second embodiment, in order to shorten the time required to form a pore of a desired size, the first and second pulse currents are We propose a method to increase the pulse width as the number of applications increases.

FIG. 18 is a time chart for executing the nanopore forming method according to the first modification of the third embodiment using the nanopore forming apparatus 100. As shown in FIG. 18, first, let the pulse width of the n-th pulse current of the first pulse current of magnitude I ₂ be t _n , and let the pulse width of the n+1-th first pulse current be t _n+1 , and t Let _n < t _n+1 . Similarly, the pulse width of the m-th second pulse current of magnitude I ₂ ′ is t _m , the pulse width of the m+1-th second pulse current is t _m+1 , and t _m <t _m+1 . As a result, the amount of stress applied to the membrane or pore by one pulse current application increases with the number of pulse current applications. Therefore, the number of times a pulse current must be applied until a pore of a desired size is formed is reduced, and as a result, the time required for a pore of a desired size to be formed is shortened.

<Modification 2 of the third embodiment>
In the second modification of the third embodiment, following the modification of the second embodiment (FIG. 15), by increasing the magnitude of the first and second pulse currents as the number of times of application increases, We propose a method to reduce the time required to form pores of a desired size.

FIG. 19 is a time chart for executing the nanopore forming method according to the second modification of the third embodiment using the nanopore forming apparatus 100. As shown in FIG. 19, the magnitude of the n-th first pulse current is I _n , the magnitude of the n+1-th first pulse current I _n+1 , and I _n <I _n+1 . However, the minimum value of the first pulse current is larger than the current _I1 . After the interelectrode current when V _th is applied reaches I ₁ or more, the pulse current is again set to a small value (second pulse current). The magnitude of the m-th second pulse current is I _m , the magnitude of the m+1-th second pulse current I _m+1 , and I _m <I _m+1 . However, the minimum value of the second pulse current is larger than the current I ₁ '.

The larger the pulse current, the higher the probability that pores will be formed during the application of the pulse current, so pores can be formed in the membrane 101 with fewer pulse applications. Also, the larger the pulse current, the more likely the pore will expand during the application of that pulse. Therefore, the number of pulses that must be applied until a pore of a desired size is formed is reduced, and as a result, the time required for a pore of a desired size to be formed is shortened.

[Fourth embodiment]
In the first to third embodiments, as a method of forming a pore of a desired size (a pore such that when the interelectrode current I is I ₁ , the interelectrode voltage is about V _th ), a pulse is applied between the electrodes. After applying the current, apply the voltage V _th and measure the inter-electrode current I at that time, and if the inter-electrode current I becomes equal to or higher than the current I ₁ (threshold value), a nanopore of the desired size is formed. He explained how to determine that the In the fourth embodiment, we propose another method of forming a pore of a desired size (a pore such that the interelectrode voltage is approximately V _th when the interelectrode current is I ₁ ) using a pulse current application method. .

<Nanopore formation method>
FIG. 20 is a flowchart of the nanopore formation method according to the fourth embodiment. In the following, each step will be explained assuming that the operator is the main person responsible for the operation, but each step can also be executed by the control computer driving each element of the control circuit.

(Step S21)
The operator drives the current source and applies a pulse current _I2 , which is larger than the current _I1 and has a pulse width _tn , between the electrodes provided across the membrane. The pulse width t _n can be set, for example, to 1 μs or more and 10 s or less, or 1 ms or more and 1 s or less.

(Step S22)
The operator drives the current source to apply a current _I1 between the electrodes provided across the membrane, and measures the potential difference between the electrodes at that time using a voltmeter.

(Step S23)
The operator determines whether the measured potential difference between the electrodes is equal to or less than the voltage V _th (threshold). If the potential difference between the electrodes is greater than the voltage V _th (No), the process moves to step S24. If the potential difference between the electrodes becomes equal to or less than the voltage V _th (Yes), the operator determines that a pore of the desired size has been formed, and ends the process.

It is desirable to set the voltage V _th to a value such that the noise current I _noise is sufficiently smaller than the nanopore current I _np .

Specifically, the voltage V _th can be set in a range of 1 mV or more and 1V or less. Alternatively, the voltage V _th can be set to be 1 mV or more and so that the electric field applied to the membrane when the voltage V _th is applied between the electrodes is 0.3 V/nm or less. Alternatively, the voltage V _th can be set to a voltage such that when a voltage is applied to the membrane in a state where no pores are formed, the current flowing through the membrane becomes 1 nA or less. Alternatively, the voltage V _th can be set to a voltage such that when a voltage is applied to the membrane in a state where no pores are formed, the current flowing through the membrane is 1/5 or less of I ₁ .

(Step S24)
The operator sets the number of times the pulse current _I2 is applied to n=n+1 and returns to step S21. In this embodiment, when the pulse width of the n-th pulse current is t _n and the pulse width of the n+1-th pulse current I ₂ is t _n+1 , t _n =t _n+1 can be satisfied.

As described above, the nanopore forming method according to the fourth embodiment uses a pulsed current I ₂ (first current) between electrodes placed across a membrane provided in an aqueous solution (electrolytic solution). , applying a current I ₁ (second current) between the electrodes and measuring the potential difference between the electrodes, and determining whether the potential difference between the electrodes is equal to or lower than the voltage V _th (predetermined threshold). If the pulse current I ₂ is set larger than the current I ₁ and the potential difference between the electrodes is larger than the voltage V _th , applying the pulse current I ₂ and determining whether the current I ₁ The application of and the measurement of the potential difference between the electrodes are repeated. Furthermore, when the potential difference between the electrodes becomes less than the voltage V _th , a pore of a desired size (a pore of a size such that the inter-electrode voltage is approximately V _th when the inter-electrode current is I ₁ ) is formed. It can be concluded that it has been formed. Thereby, pores of a desired size can be formed with a high yield. Furthermore, in this embodiment, only the current source needs to be driven, so the processing is simple.

<Nanopore formation device>
FIG. 21 is a schematic diagram showing a nanopore forming apparatus 300 for realizing the nanopore forming method according to the fourth embodiment. The nanopore forming device 300 is almost the same as the nanopore forming device 100 (FIG. 10) of the first embodiment, but the configuration of the control circuit 400 is different from the control circuit 200 of the first embodiment. Control circuit 400 includes a current source 201, a switch 204, and a voltmeter 205. Current source 201 and voltmeter 205 are arranged in parallel. By connecting switch 204 to contact A, electrodes 104 and 105 are connected to a circuit in which current source 201 and voltmeter 205 are located. By connecting switch 204 to contact B, electrodes 104 and 105 are connected to a circuit without current source 201 and voltmeter 205 (a circuit in which the electrodes are at the same potential).

FIG. 22 is a time chart for executing the nanopore forming method according to the fourth embodiment using the nanopore forming apparatus 300. As shown in FIG. 22, first, after connecting the switch 204 to the contact A, the current source 201 is driven to apply a pulse current of _I2 , which is larger than the current _I1 , between the electrodes 104 and 105. The width of the pulse current is t _n .

After the application of the pulse current is finished, by switching the switch 204 from contact A to contact B, the potential difference between the electrodes 104 and 105 becomes 0, and the potential difference generated between the electrodes 104 and 105 due to the application of the pulse current is eliminated. be able to. Thereby, when applying the current _I1 between the electrodes 104 and 105 and measuring the inter-electrode voltage at that time, the inter-electrode voltage can be measured more accurately.

Next, the switch 204 is switched from contact B to contact A again. Next, by driving the current source 201, a current _I1 is applied between the electrodes 104 and 105, and the voltage (potential difference) between the electrodes 104 and 105 at that time is measured by the voltmeter 205. The voltage between the electrodes 104 and 105 is measured after a certain period of time has elapsed since the current _I1 was applied between the electrodes 104 and 105. This fixed time is not changed depending on the number of voltage measurements.

If the voltage between the electrodes is not lower than the voltage V _th , the switch 204 is switched from contact A to contact B, and the potential difference between the electrodes is temporarily set to zero. Thereafter, the switch 204 is switched from contact B to contact A again.

Thereafter, a pulse current of _I2 , which is larger than current _I1 , is applied between electrodes 104 and 105. The width of the pulse current at that time is t _n+1 , and t _n =t _n+1 . The above procedure is repeated until the voltage between the electrodes when the current I ₁ is applied between the electrodes 104 and 105 becomes less than the voltage V _th , and the voltage between the electrodes when the current I ₁ is applied between the electrodes 104 and 105 becomes The process ends when the voltage becomes equal to or less than V _th .

In the example shown in FIG. 22, the timing at which the pulse current rises is after a certain period of time has elapsed after switching the switch 204 from contact B to contact A. The timing of switching to contact A may be simultaneous. Similarly, in the example shown in FIG. 22, the timing for switching the switch 204 from contact A to contact B is after a certain period of time has passed after the pulse current falls, but the switch 204 is switched from contact A to contact B. The switching timing and the falling timing of the pulse current may be the same.

In this embodiment as well, by setting the pulse current I ₂ to satisfy I ₁ ×1.5≦I ₂ , the possibility that the pores will not expand to the desired size after pore formation or the desired pore size may be reduced. This greatly reduces the possibility that it will take a very long time to reach the goal. Furthermore, by setting the pulse current I ₂ to satisfy I ₁ ×2≦I ₂ , these possibilities can be further reduced. Furthermore, these possibilities can be further reduced by satisfying 0.3V/nm<pulse current _I2 ×pulse width _tn ÷interelectrode capacitance (C)÷membrane thickness (nm)<2V/nm.

<Modification 1 of the fourth embodiment>
In the fourth embodiment, it has been explained that the pulse width and magnitude of the pulse current are the same in all times. On the other hand, in Modification 1 of the fourth embodiment, following the second embodiment, a pore of a desired size (a pore of a size such that the interelectrode current is approximately I1 when the interelectrode voltage is Vth) is used. In order to shorten the time required to form pores, we propose a method in which the pulse width is increased as the number of pulse current applications increases.

FIG. 23 is a time chart for executing the nanopore forming method according to the first modification of the fourth embodiment by the nanopore forming apparatus 300. As shown in FIG. 23, first, let the pulse width of the n-th pulse current of the magnitude I ₂ be t _n , and let the pulse width of the n+1-th pulse current be t _n+1 , so that t _n <t _n+1. . Since the other processes are as described above, their explanation will be omitted.

In this way, by increasing the pulse width of the pulse current with the number of times the pulse current is applied, the amount of stress applied to the membrane or pore by one application of the pulse current increases with the number of times the pulse current is applied. Therefore, the number of times a pulse current must be applied until a pore of a desired size is formed is reduced, and as a result, the time required for a pore of a desired size to be formed is shortened.

<Modification 2 of the fourth embodiment>
In the second modification of the fourth embodiment, following the modification of the second embodiment (FIG. 15), a pore of a desired size is created by increasing the magnitude of the pulse current as the number of times of application increases. We propose a method to reduce the time it takes to form.

FIG. 24 is a time chart for executing the nanopore forming method according to the second modification of the fourth embodiment by the nanopore forming apparatus 300. As shown in FIG. 24, the magnitude of the n-th pulse current is I _n , the magnitude of the n+1-th pulse current I _n+1 , and I _n <I _n+1 . However, the minimum value of the pulse current is larger than the current _I1 .

The larger the pulse current, the higher the probability that pores will be formed during the application of the pulse current, so pores can be formed in the membrane 101 with fewer pulse applications. Also, the larger the pulse current, the more likely the pore will expand during the application of that pulse. Therefore, the number of pulses that must be applied until a pore of a desired size is formed is reduced, and as a result, the time required for a pore of a desired size to be formed is shortened.

[Fifth embodiment]
In the fourth embodiment, a method for producing pores of a desired size in a membrane without pores has been described. In the fifth embodiment, we propose a method for more accurately adjusting the pores created in the membrane to a desired size. In the fifth embodiment, a pore of a desired size is a pore of a size such that when the interelectrode current is _I1 , the interelectrode voltage is approximately V _th ' (second threshold voltage). .

FIG. 25 is a flowchart of the nanopore forming method according to the fifth embodiment.

(Steps S31 to S34)
Steps S31 to S34 are almost the same as steps S21 to S24 described in the fourth embodiment, so their explanation will be omitted. However, the pulse current applied between the electrodes 104 and 105 in step S31 will be referred to as "first pulse current _I2 ." In step S34, if the inter-electrode voltage measured when the current I ₁ is applied becomes equal to or less than the voltage V _th (first threshold voltage) (Yes), the process moves to step S35.

(Step S35)
The operator drives the current source to generate a second pulse having a pulse width t m and having a magnitude greater than the current I ₁ and smaller than the first pulse current I ₂ between the electrodes provided across the _membrane . Apply current I ₂ '. The pulse width t _m can be set, for example, to 1 μs or more and 10 s or less, or 1 ms or more and 1 s or less.

(Step S36)
The operator drives the current source, applies a current _I1 between the electrodes provided with the membrane in between, and measures the inter-electrode voltage using a voltmeter.

(Step S37)
The operator determines whether the measured interelectrode voltage is equal to or lower than the voltage V _th ' (second threshold voltage). If the interelectrode voltage is greater than the voltage V _th ' (No), the process moves to step S38. If the interelectrode voltage becomes equal to or lower than the voltage V _th ' (Yes), the operator determines that the pore has reached the desired size and ends the process.

(Step S38)
The operator sets the number of applications of the second pulse current I ₂ ' to m=m+1 and returns to step S35. In this embodiment, when the pulse width of the m-th second pulse current I ₂ ′ is t _m , and the pulse width of the m+1-th second pulse current I ₂ ′ is t _m+1 , t _m = t It can be _m+1 .

The voltages V _th and V _th ' can be set to values such that the noise current I _noise is sufficiently smaller than the nanopore current I _np . Specifically, the voltages V _th and V _th ' can be set in a range of 1 mV or more and 1 V or less. Alternatively, the voltages V _th and V _th ' can be set to be 1 mV or more, and the electric field applied to the membrane when the voltages V _th and V _th ' are applied between the electrodes is 0.3 V/nm or less. . Alternatively, the voltages V _th and V _th ' can be set to voltages such that when a voltage is applied to the membrane in which no pores are formed, the current flowing through the membrane is 1 nA or less. Alternatively, the voltages V _th and V _th ' can be set to voltages such that when a voltage is applied to the membrane in which no pores are formed, the current flowing through the membrane is 1/5 or less of I ₁ .

FIG. 26 is a time chart for executing the nanopore forming method according to the fifth embodiment using the nanopore forming apparatus 300. In FIG. 26, a time chart of the switch 204 is omitted. As shown in FIG. 26, first, by driving the current source 201, a first pulse current _I2 larger than the current _I1 is applied between the electrodes 104 and 105, and then the switch 204 is switched from contact A to contact B. to temporarily reduce the potential difference between the electrodes to 0. Thereafter, the switch 204 is switched from the contact B to the contact A, and the current source 201 is driven to apply the current _I1 . After a certain period of time has elapsed since the application of the current _I1 , the voltage between the electrodes is measured by the voltmeter 205.

Repeat this, and when the interelectrode voltage when the interelectrode current is _I1 becomes less than the voltage _Vth , change the setting value of the output current of the current source 201 so that it is smaller than _I2 and larger than the current _I1 . A second pulse current I ₂ ' is applied. Thereafter, a current _I1 is applied, and the voltage between the electrodes is measured during the application. The application of the second pulse current I ₂ ′ and the voltage measurement during the application of the current I ₁ are repeated, and the process ends when the interelectrode voltage becomes equal to or lower than V _th ′.

As described above, in this embodiment, the threshold voltage is provided in two stages. That is, there are two voltages: voltage V _th and voltage V _th '. Then, from the time when the interelectrode voltage becomes equal to or lower than V _th , the pulse current is decreased from I ₂ to I ₂ ′. This makes it possible to reduce the amount by which the pore expands each time a pulse current is applied. In other words, it is possible to reduce the amount of decrease in the interelectrode voltage when _I1 is applied, which decreases each time a pulse current is applied. Therefore, it is possible to form a pore having a size closer to a desired size (a pore having a size such that the interelectrode current is approximately I ₁ when the interelectrode voltage is V _th ').

Further, pulse current I ₂ is set to satisfy I ₁ ×1.5≦I ₂ , pulse current I ₂ ′ is set to satisfy I ₁ ×1.5≦I ₂ ′, and I ₂ > By setting so that I ₂ ′>I ₁ and V _th >V _th ′ are satisfied, a pore having a size closer to a desired size can be formed.

<Modification 1 of the fifth embodiment>
In the fifth embodiment, it was explained that the pulse width and magnitude of the first pulse current are the same in all times, and the pulse width and magnitude of the second pulse current are also the same in all times. . On the other hand, in Modification 1 of the fifth embodiment, following the second embodiment, in order to shorten the time required to form a pore of a desired size, the first and second pulse currents are We propose a method to increase the pulse width as the number of applications increases.

FIG. 27 is a time chart for executing the nanopore forming method according to the first modification of the fifth embodiment by the nanopore forming apparatus 300. As shown in FIG. 27, first, let the pulse width of the n-th pulse current of the first pulse current of magnitude I ₂ be t _n , and let the pulse width of the n+1-th first pulse current be t _n+1 , and t Let _n < t _n+1 . Similarly, the pulse width of the m-th second pulse current of magnitude I ₂ ′ is t _m , the pulse width of the m+1-th second pulse current is t _m+1 , and t _m <t _m+1 . As a result, the amount of stress applied to the membrane or pore by one pulse current application increases with the number of pulse current applications. Therefore, the number of times a pulse current must be applied until a pore of a desired size is formed is reduced, and as a result, the time required for a pore of a desired size to be formed is shortened.

<Modification 2 of the fifth embodiment>
In the second modification of the fifth embodiment, following the modification of the second embodiment (FIG. 15), by increasing the magnitude of the first and second pulse currents as the number of applications increases, We propose a method to reduce the time required to form pores of a desired size.

FIG. 28 is a time chart for executing the nanopore forming method according to the second modification of the fifth embodiment using the nanopore forming apparatus 300. As shown in FIG. 28, the magnitude of the n-th first pulse current is I _n , the magnitude of the n+1-th first pulse current I _n+1 , and I _n <I _n+1 . However, the minimum value of the first pulse current is larger than the current _I1 . After the interelectrode voltage when the current _I1 is applied becomes equal to or lower than _Vth , the pulse current (second pulse current) is set to a small value again. The magnitude of the m-th second pulse current is I _m , the magnitude of the m+1-th second pulse current I _m+1 , and I _m <I _m+1 . However, the minimum value of the second pulse current is larger than the current _I1 . The second pulse current is increased as the number of applications increases until the interelectrode voltage when applying the current I ₁ becomes equal to or less than V _th '.

The larger the pulse current, the higher the probability that pores will be formed during the application of the pulse current, so pores can be formed in the membrane 101 with fewer pulse applications. Also, the larger the pulse current, the more likely the pore will expand during the application of that pulse. Therefore, the number of pulses that must be applied until a pore of a desired size is formed is reduced, and as a result, the time required for a pore of a desired size to be formed is shortened.

[Sixth embodiment]
In the first to fifth embodiments, the shape of the pulse current is described as a rectangular wave, but the shape of the pulse current does not necessarily have to be a perfect rectangular wave (constant current pulse).

FIG. 29 is a diagram showing an example of the shape of a pulse current according to the sixth embodiment. In such a case, as shown in FIG. 29, the magnitude of the pulse is defined as the maximum value in the pulse current waveform, and the pulse width is defined as the point from the rising point to the falling point of the pulse. Note that the example shown in FIG. 29 is just an example, and the waveform of the pulse current is not limited to these shapes.

[Seventh embodiment]
In the seventh embodiment, a nanopore forming apparatus will be described in which a control computer drives each element of a control circuit to execute the nanopore forming method of each embodiment.

FIG. 30 is a schematic diagram showing a nanopore forming device 500 according to the seventh embodiment. The nanopore forming apparatus 500 differs from the nanopore forming apparatus 100 of the first to third embodiments in that it further includes a control computer 600. Control computer 600 controls driving of current source 201, variable voltage 202 (voltage source), and switch 204 (switching element) of control circuit 200. Further, the control computer 600 receives input of a measurement signal from the ammeter 203. Note that the control circuit 400 described in the fourth and fifth embodiments may be used instead of the control circuit 200.

The control computer 600 includes a control section 601, an input section 602, an output section 603, and a storage section 604.

The input unit 602 is realized by, for example, an input device such as a mouse, a keyboard, and a touch panel. An operator (user) of the nanopore forming apparatus 500 can input parameters necessary for executing the nanopore forming method via the input section 602. Specifically, the operator inputs the material and thickness of the membrane 101, desired pore size, pulse current size, pulse width, switching timing of the switch 204, voltages V _th and V through the input unit 602. _th ', currents _I1 and _I1 ', etc. can be input.

The output unit 603 can be realized by, for example, an output device such as a display and a speaker. The output unit 603 displays, for example, a GUI screen for the operator to input the above parameters, the status and results of nanopore formation, etc.

The storage unit 604 stores the relationship between the size of the pore and the size of the current that flows when a certain voltage is applied to the pore, for each thickness or material of the membrane 101. The relationship between the size of the pore and the applied voltage and current is, for example, a calibration curve obtained based on measurements performed multiple times under different conditions.

The control unit 601 is realized by, for example, a processor such as a CPU or an MPU. The control unit 601 calculates an instruction value for driving each element of the control circuit 200 based on parameters input by the operator, and outputs the instruction value to each element. Each element of the control circuit 200 is driven based on the instruction value received from the control section 601. Further, when the desired pore size and the material and thickness of the membrane 101 are input by the operator, the control unit 601 reads out the calibration curve stored in the storage unit 604, and compares the calibration curve and the input pore size. Based on this, the current I ₁ or I ₁ ′ when the voltage V _th or V _th ′ is applied between the electrodes 104 and 105 can be calculated. As a result, even if the operator does not know how much current will flow when a certain voltage is applied to a pore of the desired size, the control unit 601 automatically calculates the voltage and current, so that the pore of the desired size can be drawn. Can form pores.

[Modified example]
The present disclosure is not limited to the embodiments described above, and includes various modifications. For example, the embodiments described above are described in detail to explain the present disclosure in an easy-to-understand manner, and do not necessarily include all of the configurations described. Moreover, a part of a certain embodiment can be replaced with the configuration of another embodiment. Moreover, the configuration of another embodiment can be added to the configuration of one embodiment. Further, a part of the configuration of each embodiment can be added to, deleted from, or replaced with a part of the configuration of other embodiments.

100, 300, 500... Nanopore forming device 101... Membrane 102, 103... Aqueous solution 104, 105... Electrode 106, 108... Solution inlet 107, 109... Solution outlet 110, 111... Chamber 112... Support substrate 113, 114... Seal material 200, 400...Control circuit 201...Current source 202...Variable voltage 203...Ammeter 204...Switch 205...Voltmeter 600...Control computer

Claims (17)

A method of forming pores in a membrane, the method comprising:
Applying a first current between a first electrode and a second electrode placed across the membrane provided in the electrolytic solution;
Applying a first voltage between the first electrode and the second electrode, and measuring a second current flowing between the first electrode and the second electrode at that time;
determining whether the second current is greater than or equal to a predetermined threshold;
the first current is greater than the threshold;
A hole forming method characterized in that, when the second current is smaller than the threshold, applying the first current, applying the first voltage, and measuring the second current are repeated. . The first voltage is 1 mV or more and 1 V or less, or is 1 mV or more and is a voltage that causes an electric field applied to the film to be 0.3 V/nm or less when the first voltage is applied. The pore forming method according to claim 1. The first voltage is a voltage at which, when the first voltage is applied to the membrane without holes, the current passing through the membrane becomes 1 nA or less, or 1/5 or less of the threshold value. The pore forming method according to claim 1, characterized in that: The hole forming method according to claim 1, wherein the first current is 1.5 times or more the threshold value. Hole formation according to claim 1, characterized in that 0.3V/nm<the first current×time for one application of the first current/interelectrode capacitance/thickness of the membrane<2V/nm. Method. 2. The pore forming method according to claim 1, wherein the time for one application of the first current is 1 μs or more and 10 seconds or less. 2. The pore according to claim 1, further comprising, after applying the first current, eliminating a potential difference between the electrolytic solution with the membrane sandwiched therebetween, which is caused by the application of the first current. Formation method. The first electrode and the second electrode are connected to either a first circuit having a current source or a second circuit having a voltage source and an ammeter by switching a switch,
After applying the first current, the first electrode and the second electrode are connected to the second circuit by switching the switch,
When the second current is smaller than the threshold, the first electrode and the second electrode are disconnected from the second circuit and connected to the first circuit. The pore forming method according to claim 1. A method of forming pores in a membrane, the method comprising:
Applying a first current between a first electrode and a second electrode placed across the membrane provided in the electrolytic solution;
A second current smaller than the first current is applied between the first electrode and the second electrode, and a potential difference generated between the first electrode and the second electrode at that time is measured. And,
determining whether the potential difference is less than or equal to a predetermined threshold;
A method for forming a hole, characterized in that when the potential difference is larger than the threshold, applying the first current, applying the second current, and measuring the potential difference are repeated. The threshold value is 1 mV or more and 1 V or less, or a voltage that is 1 mV or more and causes an electric field applied to the film to be 0.3 V/nm or less when the threshold voltage is applied. 9. The pore forming method according to 9. The threshold value is a voltage at which, when a voltage of the threshold value is applied to the membrane without holes, the current passing through the membrane becomes 1 nA or less, or 1/5 or less of the second current. The pore forming method according to claim 9, characterized in that: 10. The hole forming method according to claim 9, wherein the first current is 1.5 times or more as large as the second current. Hole formation according to claim 9, characterized in that 0.3V/nm<the first current×one application time of the first current/interelectrode capacitance/thickness of the membrane<2V/nm. Method. 10. The pore forming method according to claim 9, wherein the time for one application of the first current is 1 μs or more and 10 seconds or less. After the first current is applied, the first electrode and the second electrode are made to have the same potential, so that the voltage between the electrolytic solution with the membrane sandwiched therebetween, which is caused by the application of the first current, is The pore forming method according to claim 9, further comprising eliminating a potential difference. A device for forming pores in a membrane, the device comprising:
the membrane;
a first liquid tank and a second liquid tank that are arranged with the membrane in between and contain an electrolytic solution;
a first electrode disposed in the first liquid tank;
a second electrode disposed in the second liquid tank;
a first circuit having a current source;
a second circuit having a voltage source and an ammeter;
A hole forming device comprising: a switching element that connects the first electrode and the second electrode to either the first circuit or the second circuit. further comprising a control unit that controls the current source, the voltage source, and the switching element,
The control unit includes:
applying a first current between the first electrode and the second electrode by the current source;
a process of switching the switching element from the first circuit to the second circuit;
A first voltage is applied between the first electrode and the second electrode by the voltage source, and at that time, a second current flowing between the first electrode and the second electrode is converted into the current. A process of measuring with a meter,
and determining whether the second current is greater than or equal to a predetermined threshold;
the first current is greater than the threshold;
16. When the second current is smaller than the threshold, the application of the first current, the application of the first voltage, and the measurement of the second current are repeated. The pore forming device described.

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