The present disclosure relates to a nanopore formation technique for forming a nanopore in a membrane.
Nanopore sensors have been developed as biological sample analyzers for analyzing molecules, particles, DNA, proteins, and the like, for example. The nanopore sensors each include a thin film (membrane) on which pores having a size larger than or equal to a size of a biological sample (sample), such as pores having a diameter of 1 nm to 100 nm, are formed. An aqueous solution is introduced into a first solution tank and a second solution tank insulated by the membrane. The biological sample (sample) is introduced into any one of the first solution tank or the second solution tank. Voltage is applied to measure an ion current flowing between electrodes immersed in the respective solution tanks. Object characteristics and structural characteristics of the biological sample can be detected by measuring a blocked ion current flowing when the biological sample passes through the pores.
The nanopore sensors are classified into two types including one type based on a biological membrane and the other type based on a solid-state membrane. The nanopore sensor using the biological membrane includes pores formed by floating proteins having pores of a nanometer size on a lipid bilayer membrane. In contrast, the nanopore sensor using the solid-state nanopore includes a membrane made of a high dynamic strength material such as silicon nitride (SiN), and pores of a nanometer size that are manufactured by electron beam irradiation to the membrane, a chemical etching technique, or a dielectric breakdown technique in which a voltage is applied to the membrane to form pores. The dielectric breakdown technique causes a potential difference in the membrane using an external power supply or the like to apply stress to the membrane, thereby forming a nanopore by dielectric breakdown. The dielectric breakdown technique has advantages such as an inexpensive nanopore manufacturing cost, user friendliness, on-site processing (processing at a user's place of use), and ability to form fresh nanopores because nanopores can be formed on a membrane immediately before a target sample is measured.
For example, PTL 1 or NPL 1 discloses formation of nanopores on a SiN membrane using a neutral pH aqueous solution (pH 7-7.5). The introduced aqueous solution may sometimes not come into contact with a surface of a dry membrane because the surface of the dry membrane becomes hydrophobic. Thus, a yield of nanopore formation may be deteriorated. The reason why a solid-state membrane becomes hydrophobic due to surrounding environment is considered to be that its surface is oxidized or/and unnecessary organic substances adhere to the surface. Thus, a laboratory is required to usually perform a treatment for hydrophilizing the membrane before use. In general, a laboratory performs hydrophilicity processing by piranha processing or plasma processing. However, when the nanopore sensor is commercialized, causing an end user to perform these dangerous processes is not realistic, and thus another alternative technique is required.
For example, PTL 2 discloses introduction of a method for storing a membrane in an aqueous solution at specific conditions such as the amount of water, temperature, and salinity. PTL 3 discloses introduction of a method for storing a membrane by attaching a hydrophilic surface and a protective layer containing a soluble substance to the membrane. PTL 4 discloses introduction of an electrowetting on dielectric (EWOD) method in which droplets of an aqueous solution are moved to a position of a membrane by applying high voltage to control hydrophobicity and a hydrophilic property, and this method also requires plasma treatment of a surface of the membrane. PTL 5 discloses introduction of a nanopore production method based on a pulsed laser in which piranha treatment is performed on a sample before measurement to achieve hydrophilicity.
PTL 1 and NPL 1, however, cause a low yield of nanopore formation due to a dry membrane surface having hydrophobic characteristics although the dielectric breakdown is performed using a neutral pH aqueous solution without the piranha treatment or the plasma treatment. Additionally, requiring the piranha treatment or the plasma treatment to be performed at a user's place of use (on-site) causes problems that a user is burdened with performing processing and is unfavorable to safety, and thus is not realistic. Even when the alternative preservation techniques according to PTLS 2 and 3 are used, a preservation process itself may cause a burden on the user.
By contrast, an alkaline aqueous solution or a high pH aqueous solution has low surface tension characteristics, and so can wash away unnecessary substances that enter a position of a membrane and adhere to a surface of the membrane. NPL 2 describes nanopores that are formed on a membrane of about 20 nm using a high pH aqueous solution as described above. However, the high pH aqueous solution is used for a chemical etching process, so that a sub-nanometers to nanometers size of the nanopores to be formed cannot be controlled, and thus the membrane may be deteriorated. Precise control of nanopore size on the order of sub-nanometers is considered to be important in performing practical biological analysis such as DNA sequencing. In view of such a situation, the present disclosure proposes a technique for forming a nanopore having a high yield and an appropriate size.
To solve the above problems, the present disclosure proposes a nanopore formation method for forming a nanopore in a membrane, the method including: introducing an aqueous solution at a first pH into a first solution tank and a second solution tank insulated by the membrane; securing hydrophilicity of a surface of the membrane; replacing the aqueous solution at the first pH in the first solution tank and the second solution tank with an aqueous solution at a second pH lower than the first pH after the hydrophilicity of the surface of the membrane is secured; and immersing an electrode in each of the first solution tank and the second solution tank containing the aqueous solution at the second pH to cause dielectric breakdown and form a nanopore in the membrane.
Further features related to the present disclosure will become apparent from the description herein and the accompanying drawings. Aspects of the present disclosure are achieved and implemented by elements, combinations of various elements, and aspects of the following detailed description and the scope of claims appended.
According to the technique of the present disclosure, it becomes possible to form a nanopore having a high yield and an appropriate size.
A membrane in the present embodiment is not restricted in material, thickness, size, and number. There are no restrictions in composition on a high pH aqueous solution, a neutral pH aqueous solution used for nanopore formation, and an aqueous solution used for sample measurement, respectively in the present embodiment. A dielectric breakdown technique used for nanopore formation is merely an example. Various forms of dielectric breakdown technique can be applied. Although a capacitance noise measurement method is used when measuring RMS noise in the present embodiment, the present invention is not limited thereto.
(i) A nanopore sensing system in the present embodiment includes a dry membrane that is attached to a flow cell (a part of the nanopore sensing system) before a nanopore is formed to insulate a first solution tank and a second solution tank of the flow cell. A high pH aqueous solution (first pH aqueous solution) is introduced (filled) into each solution tank. An electrode is immersed in the solution in each bath and connected to an electric device unit capable of supplying a voltage or current. Before a nanopore is formed, capacitance noise is first measured in an unloaded state and a loaded state (a capacitance noise measurement process). It is checked whether a membrane surface becomes hydrophilic, and an aqueous solution is in contact with the membrane surface. When hydrophilicity of the membrane surface has been checked, the high pH aqueous solution in each solution tank is rapidly replaced by an aqueous solution at a lower pH (second pH aqueous solution: neutral pH aqueous solution) (aqueous solution replacement process). This is because the membrane is likely to be decomposed by a chemical etching process using a high pH aqueous solution. Subsequently, the electrode is immersed in each solution tank, and a voltage or a current is supplied by the electric device unit. As a result, dielectric breakdown occurs, and a nanopore is formed in the membrane.
(ii) In the capacitance noise measurement process, a DC voltage is applied between the two electrodes. Then, root mean square (RMS) noise or a noise of the electric device unit is monitored in a state without a load (a state where the electrode is not inserted into each of the solution tanks insulated by the membrane) and a state with a load (a state where the electrode is inserted into each of the solution tanks insulated by the membrane). Here, the DC voltage can be made lower than dielectric breakdown induction voltage of the membrane. For example, when a membrane having a thickness of 5 nm is used, the membrane has a dielectric breakdown voltage of 5 V, and thus capacitance noise is checked by applying a DC voltage from −2.5 V to 2.5 V. As described above, when a voltage lower than the dielectric breakdown induction voltage is applied in the capacitance noise measurement process, unnecessary dielectric breakdown can be prevented from occurring during the process.
In the present embodiment, when the capacitance noise with a load (RMS noise with a load) is several times (at least twice or more) higher than the capacitance noise without a load (RMS noise without a load), it is determined that the membrane surface becomes hydrophilic or the aqueous solution is in contact with the membrane surface. In contrast, when the RMS noise with a load is equal to or less than the RMS noise without a load, the aqueous solution may not be in contact with the membrane surface. Thus, the high pH aqueous solution (first pH aqueous solution) is taken out from each solution tank and reintroduced (filled). When the first pH aqueous solution is reintroduced, pH of the first pH aqueous solution may be further increased. The contact between the first pH aqueous solution and the membrane surface may be ensured by moving the first pH aqueous solution back and forth on the membrane surface (e.g., pipetting processing).
(iii) The aqueous solution replacement process is performed to take out the first pH aqueous solution (high pH aqueous solution) from each solution tank and to introduce a second pH aqueous solution (e.g., a neutral pH aqueous solution) at a pH lower than the first pH into each solution tank. This process may be performed several times to cause the aqueous solution in each bath to be reliably neutral. When the aqueous solution replacement process is performed, it may be checked whether the pH of the aqueous solution in each solution tank is neutral, for example.
(iv) A nanopore of a desired size can be formed on the membrane by causing dielectric breakdown. A sample can be introduced into any one of two solution tanks. When a voltage is applied between the electrodes of the respective solution tanks or when a current flows between the electrodes, an ion current is measured. A target molecule or particle can be detected by measuring the ion current (blockage current) when the target molecule or particle passes through a nanopore.
(v) A low utilization rate of a solid-state nanopore sensor due to hydrophobic properties of the dry membrane is overcome by introducing a high pH aqueous solution, as described above. After checking by measuring the capacitance noise that the aqueous solution is in contact with the membrane surface, the size of the nanopore is effectively controlled by rapidly replacing the aqueous solution with a neutral pH aqueous solution for nanopore production by the dielectric breakdown technique. Additionally, a burden of using advanced preservation processing of the membrane is also avoided.
First, capacitance noise (RMS noise) is measured while the 1M KCl pH 12.7 aqueous solutions 106 and 112 are contained in the first solution tank 103 and the second solution tank 110, respectively, as illustrated in
After the aqueous solutions are replaced, the electric device unit 107 applies a voltage or current based on the dielectric breakdown technique to enable a nanopore 113 of a desired size to be formed in the membrane 101 (pore size is controllable).
<Details of Processing from Forming Nanopore to Measuring Sample>
(i-1) Step 202
First, an operator prepares a dry membrane 101.
(i-2) Step 203
Next, the operator attaches the dry membrane 101 to a flow cell. Here, the flow cell corresponds to a component part including the first solution tank 103, the second solution tank 110, and the liquid sealing gaskets 102 and 111 in the nanopore sensing system 100 (see
(i-3) Step 204
The operator introduces (fills) an aqueous solution at a high pH (higher than pH 8), or an aqueous solution at a first pH, such as an aqueous solution at a pH of 1M KCl of 12.7, into the first solution tank 103 and the second solution tank 110 of the flow cell.
The hydrophilicity checking processing (step 211) includes the steps of: applying a DC voltage Vc between electrodes (step 212); measuring RMS noise without inserting an electrode into the flow cell (the first solution tank 103 and the second solution tank 110) (RMS noise measurement without a load: step 213); inserting an electrode into the flow cell (step 216); and measuring RMS noise (RMS noise measurement with a load: step 214).
(ii-1) Step 212
The operator applies the DC voltage (Vc) between the Ag/AgCl electrodes 104 and 109 using the electric device unit 107. At this stage, the Ag/AgCl electrodes 104 and 109 are not immersed in the aqueous solutions of the first solution tank 103 and the second solution tank 110, respectively. At this time, a low voltage of 10 V or less is applied, for example. This is to prevent undesired dielectric breakdown from occurring in step 214.
(ii-2) Step 213
The operator measures root mean square (RMS) noise (RMS noise σno without a load) of the current in a state where the Ag/AgCl electrodes 104 and 109 are not immersed in the aqueous solution (aqueous solution at the first pH).
(ii-3) Step 216
The operator immerses the Ag/AgCl electrodes 104 and 109 in the aqueous solution (aqueous solution at the first pH).
(ii-4) Step 214
Subsequently, the operator measures root mean square (RMS) noise (RMS noise σw with a load) of a current in a state where the Ag/AgCl electrodes 104 and 109 are immersed in an aqueous solution (aqueous solution at a first pH), that is in a state where the electrodes 104 and 109 are inserted into the first solution tank 103 and the second solution tank 110, respectively.
(ii-5) Step 215
The operator compares the RMS noise σno without a load and the RMS noise σw with a load. Specifically, it is determined whether the RMS noise σw is several times (e.g., twice) larger than the RMS noise σno without a load. When the RMS noise σw with a load is larger than the RMS noise σno without a load (Yes in step 215), it is confirmed that the surface of the membrane 101 has hydrophilicity (a hydrophilic property: a state in which the surface of the membrane 101 is completely in contact with the aqueous solution), and that the hydrophilicity checking processing (step 211) ends. Then, the processing proceeds to the nanopore forming processing S221. When the RMS noise σw with a load is equal to or less than the RMS noise σno without a load (No in step 215), the hydrophilicity of the surface of the membrane 101 is not ensured, and then the processing returns to step 204. In this case, the aqueous solutions (aqueous solution at the first pH, such as an aqueous solution of 1M KCl pH 12.7) in the first solution tank 103 and the second solution tank 110 are discharged in step 204, and the first solution tank 103 and the second solution tank 110 are filled with a new aqueous solution at the first pH. Then, the hydrophilicity checking processing (step 211) is performed again. When even the hydrophilicity checking processing performed multiple times (e.g., five times) fails to check hydrophilicity of the surface of the membrane 101 during the hydrophilicity checking processing performed again, the pH of the aqueous solution may be increased and set lower than 14.
Although the operator compares the RMS noise σno without a load and the RMS noise σw with a load in the above description, a computer (not illustrated) may be connected to the nanopore sensing system 100 to allow the computer to perform processing of the comparison. At this time, when the RMS noise σw with a load is larger than the RMS noise σno without a load, the computer controls a display device to display an instruction for transition of the processing to step 221 on a screen (not illustrated) of the display device, thereby enabling prompting the operator to perform the next nanopore forming processing. Then, when the RMS noise σw with a load is equal to or less than the RMS noise σno without a load, the computer contorols a display device to display an instruction for transition of the processing to step 204 on the screen (not illustrated) of the display device, thereby enabling prompting the operator to perform the hydrophilicity checking processing again. Alternatively, the RMS noise σno without a load may be measured in advance and stored in a memory (not illustrated) to read out the RMS noise σno without a load from the memory when the hydrophilicity checking processing is performed, thereby comparing the RMS noise σno with the RMS noise σw with a load.
(iii) Step 221
(iii-1) Step 222
The operator discharges the aqueous solution at the first pH with which the first solution tank 103 and the second solution tank 110 are filled. Instead, the operator fills an aqueous solution at the second pH lower than the first pH, such as a neutral aqueous solution, more particularly an aqueous solution of 1M KCl pH 7.5 (replacement of the aqueous solution).
(iii-2) Step 223
The operator operates the electric device unit 107 to apply a voltage between the electrodes 104 and 109 or allows a current to flow therebetween to cause dielectric breakdown, and forms the nanopore 113 having a desired size in the membrane 101 as illustrated in
As in the nanopore forming processing (step 221), the first solution tank 103 and the second solution tank 110 in the sample measuring processing are filled with the aqueous solution at the second pH, such as a neutral aqueous solution, more specifically an aqueous solution of 1M KCl pH 7.5, in which a type of the aqueous solution (such as KCl) rather than pH varies depending on a type of sample to be measured and introduced.
(iv-1) Step 232
As illustrated in
(iv-2) Step 233
The operator operates the electric device unit 107 to apply a low voltage, such as a voltage lower than 20 V, and more specifically lower than 2 V, between the electrodes 104 and 109 to measure an ion current (blockage current) when the sample 120 passes through the nanopore.
The operator uses the electric device unit 107 to apply a pulse voltage Vp1 between the Ag/AgCl electrodes 104 and 109 immersed in the first solution tank 103 and the second solution tank 110, respectively, for at least one period (step 241). Although the pulse voltage Vp1 varies in magnitude depending on thickness and material of the membrane 101, a pulse voltage of 4 V to 5 V is applied to a SiN membrane with a thickness of 5 nm, for example. The pulse voltage Vp1 serves to apply stress to the membrane 101.
Subsequently, the operator operates the electric device unit 107 to change the applied voltage from the pulse voltage Vp1 to a predetermined low voltage Vm1, and then measures a current Im1 flowing at the low voltage Vm1 (step 242). Here, the low voltage Vm1 is set lower than a value of the pulse voltage Vp1 to such an extent that dielectric breakdown does not occur during processing in step 242 or a nanopore already formed does not expand. For example, when a SiN membrane having a thickness of 5 nm is used as the membrane 101, the low voltage Vm1 is set less than 1 V.
The operator compares the measured current Im1 with a threshold current Ith1 (step 253). When the measured current Im1 is larger than the threshold current Ith1 (Yes in step 243), it is determined that the nanopore 113 desired (see
The operator uses the electric device unit 107 to apply a constant voltage Vdc between the Ag/AgCl electrodes 104 and 109 immersed in the first solution tank 103 and the second solution tank 110, respectively, for a predetermined time (step 251). Although the constant voltage Vdc varies in magnitude depending on thickness and material of the membrane 101, a constant voltage of 16 V to 20 V can be applied to a SiN membrane with a thickness of 20 nm, for example. Application time of the constant voltage is less than 10 minutes including application time in the next step 252, for example.
Subsequently, while applying the constant voltage Vdc, the operator measures a current Im2 flowing at that moment (step 252).
Then, the operator compares the measured current Im2 with a threshold current Ith2, and checks whether the nanopore 113 desired is formed in the membrane 101 (step 253). When the measured current Im2 is larger than the threshold current Ith2 (Yes in step 253), it is determined that the nanopore 113 desired (see
(iii) Example of Causing Pulse Current to Flow:
The operator uses the electric device unit 107 to cause a pulse current Ip1 to flow between the Ag/AgCl electrodes 104 and 109 immersed in the first solution tank 103 and the second solution tank 110, respectively, for at least one period (step 261). The pulse current Ip1 varies in magnitude depending on thickness and material of the membrane 101.
Subsequently, the operator operates the electric device unit 107 to apply a predetermined low voltage Vm2 between the Ag/AgCl electrodes 104 and 109 immersed in the first solution tank 103 and the second solution tank 110, respectively, and then measures a current Im3 flowing at the low voltage Vm2 (step 262). Here, the low voltage Vm2 is set low to such an extent that dielectric breakdown does not occur during processing in step 262 or a nanopore already formed does not expand. For example, when a SiN membrane having a thickness of 5 nm is used as the membrane 101, the low voltage Vm1 is set less than 1 V.
The operator compares the measured current Im3 with a threshold current Ith3 (step 263). When the measured current Im3 is larger than the threshold current Ith3 (Yes in step 263), it is determined that the nanopore 113 desired (see
The operator uses the electric device unit 107 to cause a constant current Idc to flow between the Ag/AgCl electrodes 104 and 109 immersed in the first solution tank 103 and the second solution tank 110, respectively, for a predetermined time (step 271). The constant current Idc varies in magnitude depending on thickness and material of the membrane 101. While causing the constant current Idc to flow, the operator measures the voltage Vm3 at that time (step 272).
Then, the operator compares the measured voltage Vm3 with a threshold voltage Vtn, and checks whether the nanopore 113 desired is formed in the membrane 101 (step 273). When the measured voltage Vm3 is larger than the threshold voltage Vth (Yes in step 273), it is determined that the nanopore 113 desired (see
Filing Document | Filing Date | Country | Kind |
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PCT/JP2021/025047 | 7/1/2021 | WO |