Patent Publication Number: US-2022214326-A1

Title: Biopolymer analysis device, biopolymer analysis equipment, and biopolymer analysis method

Description:
TECHNICAL FIELD 
     The present disclosure relates to a biopolymer analysis device, biopolymer analysis equipment, and a biopolymer analysis method. 
     BACKGROUND ART 
     In a nanopore device, a pore having a diameter of several A to several nm (hereinafter, referred to as a nanopore) is provided in a thin film having a thickness of several A to several tens of nm, an electrolyte solution is brought into contact with both sides of the thin film and a potential difference is generated between both ends of the thin film. Thus, the electrolyte solution can pass through the nanopore. At this time, when an object to be measured in the electrolyte solution passes through the nanopore, since electrical characteristics, particularly a resistance value, of a peripheral portion of the nanopore change, the object to be measured can be detected by detecting the change in the electrical characteristics. When the object to be measured is a biopolymer, the electrical characteristics of the peripheral portion of the nanopore changes in a pattern shape according to a monomer sequence pattern of the biopolymer. In recent years, a method for analyzing a monomer sequence of a biopolymer by using such a change has been actively studied. 
     Among these studies, the analysis of the monomer sequence based on the principle that the amount of change in ion current observed when the biopolymer passes through the nanopore varies depending on monomer species has been expected. Since the analysis accuracy of the monomer sequence is decided by the amount of change in the ion current, it is desirable that a difference in the amount of ion current between the monomers is large. Such an analysis method can directly read the biopolymer without requiring a chemical operation involving fragmentation of the biopolymer unlike the method in the related art. The nanopore device is used as a DNA base sequence analysis system (DNA sequencer) when the biopolymer is DNA, and is used as an amino acid sequence analysis system (amino acid sequencer) when the biopolymer is protein. These systems are expected as systems capable of decoding a sequence length much longer than in the related art. 
     In particular, research and development to put the nanopore into practical use as the DNA sequencer by using a blockade current system is active. The blockade current is a decrease amount of the ion current due to a decrease in an effective cross-sectional area through which ions can pass since the biopolymer blocks the nanopore when the biopolymer passes through the nanopore. 
     As the nanopore device, there are two types of nanopore devices of a bio-nanopore using a protein having a pore at a center embedded in a lipid bilayer membrane and a solid-state nanopore in which a pore is processed in an insulating thin film formed by a semiconductor processing process. In the bio-nanopore, the amount of change in ion current is measured by using a pore (diameter 1.2 nm and thickness 0.6 nm) of a modified protein (such as  Mycobacterium smegmatis  porin A (MspA)) embedded in the lipid bilayer membrane as a biopolymer detection unit. 
     On the other hand, in the solid-state nanopore, a structure in which a nanopore is formed in a thin film of silicon nitride (SiN) which is a semiconductor material or a thin film including a monolayer such as a graphene or molybdenum disulfide is used as the nanopore device. In a biopolymer analysis method using the solid-state nanopore, there have been reported so far on measurement of the amounts of blockade currents of an adenine base, a cytosine base, a thymine base, and a guanine base of a homopolymer (NPL 1 and NPL 2). 
     In the measurement using the nanopore device, there are the following three problems. A first problem is that individual independent channels need to be insulated from each other without leakage of a current between the individual independent channels in order to realize an integrated nanopore device having arrayed parallel channels. When the individual independent channels are not insulated, the individual independent channels interfere with each other, and accurate measurement cannot be performed. Thus, it becomes difficult to perform independent measurement of each channel. 
     As a second problem, when a sample is depleted during measurement and a measurement throughput is reduced, or when it is desired to measure another sample after a certain sample is sufficiently measured, an effective continuous measurement time needs to be extended by performing smooth sample supply or sample replacement. 
     As a third problem, when a biomolecule represented by DNA is measured, since a sample collected from a living body is valuable and it is desirable to collect only a small volume, it is necessary to perform measurement even with a small solution volume (small DNA input amount). 
     In PTL 1, in order to realize an integrated nanopore device using a lipid bilayer membrane and a bio-nanopore, the following method has been attempted. A water-repellent liquid (oil) and an aqueous solution containing a material constituting a lipid bilayer membrane are alternately poured into a resin flow cell having a plurality of parallel wells, and thus, individual droplet portions are spontaneously formed at a bottom portion of each parallel well, and a common solution portion is spontaneously formed at a well ceiling portion. The integration is realized by spontaneously forming the lipid bilayer membrane at an interface between each individual droplet portion and the common solution portion and electrically embedding the bio-nanopore in the membrane. 
     Unlike the lipid bilayer membrane using self-assembly of the bio-nanopore, in the solid-state nanopore device, since a solid inorganic thin film made of an inorganic material in advance is used, the method as in PTL 1 cannot be applied, and another approach is required to realize the integration. In NPL 3, a method for forming five parallel channels by dividing one inorganic thin film into separate sections by using a microchannel has been attempted. 
     In NPL 4, a method for realizing parallelization by combining an O-ring of insulating rubber and a resin flow cell for a device having 16 independent thin films has been attempted. 
     In PTL 2, in order to realize a parallelized solid-state nanopore device having a high degree of integration, a method for using a water-repellent liquid (oil) as an insulator between independent channels has been attempted. Such a water-repellent liquid is realized by a liquid feeding mechanism using a channel. PTL 3 describes a method for providing an insulating film such as a photosensitive resin as an insulating partition wall between independent channels. Such an insulating film is realized by a liquid feeding mechanism using a pressure bonding method. 
     As described above, what is common to the integrated nanopore devices is that, a common solution tank is provided on one side of the thin film, and a plurality of independent individual solution tanks is provided on the other side. Such a configuration is a basic structure in the integrated nanopore device. 
     CITATION LIST 
     Patent Literature 
     
         
         PTL 1: WO2014/064443A 
         PTL 2: JP 6062569 B 
         PTL 3: JP 2018-96688 A 
       
    
     Non-Patent Literature 
     
         
         NPL 1: Feng J., et al., Identification of single nucleotides in MoS 2  nanopores. Nat. Nanotechnol. 10, 1070-1076 (2015). 
         NPL 2: Goto Y., et al., Identification of four single-stranded DNA homopolymers with a solid-state nanopore in alkaline CsCl solution. Nanoscale 10, 20844-20850 (2018). 
         NPL 3: Tahvildari R., et al., Integrating nanopore sensors within microfluidic channel arrays using controlled breakdown. Lab on a Chip 15, 1407-1411 (2015). 
         NPL 4: Yanagi I., et al, Multichannel detection of ionic currents through two nanopores fabricated on integrated Si 3 N 4  membranes. Lab on a Chip 16, 3340-3350 (2016). 
       
    
     SUMMARY OF INVENTION 
     Technical Problem 
     However, in the integrated solid-state nanopore system of the related art, it is difficult to achieve both collective injection of solutions into a plurality of independent individual solution tanks and solution (sample) replacement in the individual solution tanks while maintaining insulation between the channels. Although it is easy to replace the solutions by using a channel such as a flow cell, a special jig or a liquid feeding device such as a pump is required to collectively inject the solutions into the individual solution tank, and the device becomes complicated. This problem is remarkable when the degree of integration increases and the channel is minute. 
     Since the individual solution tank formed by the pressure bonding method as in PTL 3 is a closed space, it is difficult to replace the solutions in the first place. 
     In the method of the related art, since a solution volume larger than a solution volume of the individual solution tank is required to arrange the solutions in the individual solution tanks, there is also a problem that it is difficult to measure the sample with a small solution volume. 
     Therefore, the present disclosure provides a technology for achieving both automatic collective injection of solutions into a plurality of individual solution tanks and automatic replacement of the solutions in the individual solution tanks while maintaining insulation between parallel channels. 
     Solution to Problem 
     In order to solve the above problems, a biopolymer analysis device of the present disclosure includes an insulating thin film that is made of an inorganic material, a first liquid tank and a second liquid tank that are separated by the thin film, a plurality of first electrodes that is arranged in the first liquid tank, and a second electrode that is disposed in the second liquid tank. A water-repellent liquid and a plurality of liquid droplets are introduced into the first liquid tank, the plurality of first electrodes is configured to be able to convey the plurality of droplets introduced into the first liquid tank by electro wetting on dielectric by applying a certain voltage, and the plurality of droplets is conveyed to portions coming into contact with the plurality of first electrodes, and is insulated from each other by the water-repellent liquid. 
     Further features related to the present disclosure will be apparent from the description of the present specification and the accompanying drawings. The aspects of the present disclosure are achieved and realized by elements, combinations of various elements, the following detailed description, and aspects of the appended claims. 
     The description of the present specification is merely a typical example, and does not limit the scope of claims or application examples of the present disclosure in any sense. 
     Advantageous Effects of Invention 
     According to the present disclosure, it is possible to achieve both automatic collective injection of solutions into a plurality of individual solution tanks and automatic replacement of the solutions in the individual solution tanks while maintaining insulation between parallel channels. 
     Other objects, configurations, and effects will be made apparent from the following descriptions. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a schematic diagram illustrating a biopolymer analysis device with a single channel according to a reference example. 
         FIG. 2  is a schematic diagram illustrating a biopolymer analysis device with parallel channels according to a reference example. 
         FIG. 3A  is a schematic diagram illustrating a biopolymer analysis device according to a first embodiment. 
         FIG. 3B  is a schematic diagram illustrating the biopolymer analysis device after a nanopore is opened. 
         FIG. 4  is a schematic diagram illustrating another biopolymer analysis device according to the first embodiment. 
         FIG. 5  is a schematic diagram illustrating another biopolymer analysis device according to the first embodiment. 
         FIG. 6  is a flowchart illustrating a biopolymer analysis method according to the first embodiment. 
         FIG. 7  is a schematic diagram illustrating a biopolymer analysis device according to a second embodiment. 
         FIG. 8A  is a top view of a first liquid tank of the biopolymer analysis device according to the second embodiment. 
         FIG. 8B  is a top view illustrating a scene in which droplets are conveyed. 
         FIG. 8C  is a top view illustrating a state in which all the droplets are arranged at desired positions. 
         FIG. 9  is a schematic diagram illustrating a biopolymer analysis device according to a third embodiment. 
         FIG. 10A  is a schematic diagram illustrating a state in which a water-repellent liquid remains on a surface of a thin film. 
         FIG. 10B  is a schematic diagram illustrating a structure of a sacrificial layer according to a fourth embodiment. 
         FIG. 10C  is a schematic diagram illustrating another biopolymer analysis device according to the fourth embodiment. 
         FIG. 11  is a schematic diagram illustrating a biopolymer analysis device according to a fifth embodiment. 
         FIG. 12  is a schematic diagram illustrating another biopolymer analysis device according to the fifth embodiment. 
         FIG. 13  is a schematic diagram illustrating a biopolymer analysis device according to a sixth embodiment. 
         FIG. 14A  is a schematic diagram illustrating a biopolymer analysis device according to a seventh embodiment. 
         FIG. 14B  is a schematic diagram illustrating the biopolymer analysis device according to the seventh embodiment. 
         FIG. 15  is a schematic diagram illustrating biopolymer analysis equipment according to an eighth embodiment. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. Although the accompanying drawings illustrate specific embodiments based on the principles of the present disclosure, the drawings are provided for understanding the present disclosure, and are not used for restrictively interpreting the present disclosure. 
     Biopolymer analysis devices have different configurations depending on a method for introducing biopolymers into a nanopore. In the present specification, a method for introducing biopolymers into a nanopore by electrophoresis will be described as an example. Here, the biopolymer refers to DNA or RNA having a nucleic acid as a monomer, or a protein or a polypeptide having an amino acid as a monomer. 
     REFERENCE EXAMPLES 
       FIG. 1  is a schematic diagram illustrating a biopolymer analysis device  100  having a single nanopore channel according to a reference example. As illustrated in  FIG. 1 , the biopolymer analysis device  100  includes a thin film  102  having a nanopore  101 , a first liquid tank  104 A and a second liquid tank  104 B storing an electrolyte solution  103 , and electrodes  105 A and  105 B. 
     The electrodes  105 A and  105 B are connected to an ammeter  106  and a power supply  107 . A voltage is applied to the electrode  105 A and the electrode  105 B by the power supply  107 . The application of the voltage by the power supply  107  is controlled by a computer  108 . 
     The ammeter  106  measures an ion current (blockade current) flowing between the electrode  105 A and the electrode  105 B. Although not illustrated, the ammeter  106  includes an amplifier that amplifies the current flowing between the electrodes  105 A and  105 B and an analog-to-digital converter. The ammeter  106  is connected to the computer  108 , and the analog-to-digital converter outputs, as a digital signal, a value of the detected ion current to the computer  108 . 
     The computer  108  acquires monomer sequence information of biopolymers  1  based on the value of the ion current (blockade current). 
       FIG. 2  is a schematic diagram illustrating a biopolymer analysis device  200  as an array device having parallel nanopore channels according to a reference example. The array device refers to a device including a plurality of individual solution tanks partitioned by partition walls. As illustrated in  FIG. 2 , the biopolymer analysis device  200  is different from the biopolymer analysis device  100  of  FIG. 1  in that a plurality of second liquid tanks  104 B electrically insulated by a tapered layer  102 B as a partition wall is provided, and electrodes  105 B are provided in the plurality of second liquid tanks  104 B, respectively. 
     As described above, the first liquid tank  104 A is a common solution tank, the second liquid tanks  104 B are a plurality of individual solution tanks, and a plurality of independent channels is formed. The electrode  105 A is a common electrode, and the electrodes  105 B are individual electrodes. 
     First Embodiment 
     &lt;Configuration Example of Biopolymer Analysis Device&gt; 
       FIG. 3A  is a schematic diagram illustrating a biopolymer analysis device  300  according to a first embodiment. As illustrated in  FIG. 3A , the biopolymer analysis device  300  is a solid-state nanopore device, and includes a thin film  102  made of an inorganic material, a first liquid tank  104 A, a second liquid tank  104 B, a common electrode  105  (second electrode), and a substrate  113  having a plurality of individual electrodes  112  (a plurality of first electrodes). 
     The material of the thin film  102  is an insulating inorganic material that can be formed by a semiconductor microfabrication technique. Examples of the material of the thin film  102  include silicon nitride (SiN), silicon oxide (SiO 2 ), silicon oxynitride (SiON), hafnium oxide (HfO 2 ), molybdenum disulfide (MoS 2 ), a graphene, and the like. A thickness of the thin film  102  can be, for example, from 1 Å to 200 nm, optionally from 1 Å to 100 nm or from 1 Å to 50 nm, for example about 5 nm. 
     Although not illustrated, the common electrode  105  can be connected to the ammeter  106 , the power supply  107 , and the computer  108  (controller) illustrated in  FIGS. 1 and 2  via wirings, and the plurality of individual electrodes  112  can be connected to the ammeter  106 , the power supply  107 , and the computer  108  via wirings inside the substrate  113 . 
     As will be described later, the computer  108  controls application of a voltage to the plurality of individual electrodes  112  and the common electrode  105  by the power supply  107 . The computer  108  applies a voltage between the plurality of individual electrodes  112  or between each individual electrode  112  and the common electrode  105 , and determines positions of droplets  110 , whether or not a leak occurs between the droplets  110 , or whether or not nanopores are formed in the thin film  102  based on electrical characteristics such as a measured current value. The computer  108  includes a storage (not illustrated), and stores the measured current value or the determination result in the storage. 
     The plurality of individual electrodes  112  is embedded in the substrate  113 . The substrate  113  constitutes a part of the first liquid tank  104 A. The material of the substrate  113  may be any material as long as circuit wirings can be mounted, and for example, a PWB substrate or a PCB substrate such as glass epoxy resin is used. Alternatively, the substrate  113  may be a transparent substrate such as a glass substrate. 
     A plurality of droplets  110  and a water-repellent liquid  111  are introduced into the first liquid tank  104 A. Each droplet  110  is electrically insulated from the adjacent droplet  110  by the water-repellent liquid  111 , and the droplets are independent of each other. The plurality of droplets  110  comes into contact with the individual electrode  112 , respectively, and thus, an electrical operation such as application of a voltage can be performed on each droplet  110 . 
     A certain voltage is applied between the adjacent individual electrodes  112 , and thus, the individual electrodes  112  convey the droplets  110  to desired positions by electro wetting on dielectric (EWOD).  FIG. 3A  illustrates a state in which the droplets  110  are conveyed to positions coming into contact with the individual electrodes  112 , and the droplets  110  are separated from each other by the water-repellent liquid  111  and are insulated from each other. Accordingly, the plurality of individual solution tanks (the plurality of channels) is formed. 
     Application of an EWOD conveying voltage (certain voltage) for operating the individual electrodes  112  as EWOD electrodes is controlled by the computer  108 . The EWOD conveying voltage can be set to, for example, 0 to 100V, and is typically set in a range of 10 to 50V. This voltage value changes every time depending on a diameter and a viscosity of the droplet  110 , a contact angle between the droplet  110 , the water-repellent liquid  111 , and the individual electrode  112 , an electrode size, or the like, and thus, the voltage value is appropriately adjusted. 
     The individual electrode  112  is also used to open the nanopores  101  or measure the ion current by applying a voltage between the individual electrode  112  and the common electrode  105 . 
     An electrolyte solution  103  as a common solution is introduced into the second liquid tank  104 B. The common electrode  105  is disposed so as to come into contact with the electrolyte solution  103 . Here, the plurality of droplets  110  and the electrolyte solution  103  are aqueous solutions containing an electrolyte, and may contain biopolymers to be analyzed. 
     The volume of the electrolyte solutions  103  can be on the order of microliters or milliliters. The volume of the droplets  110  can be on the order of nanoliter or microliter. 
     The first liquid tank  104 A and the second liquid tank  104 B that store a measurement solution coming into contact with the thin film  102  can be appropriately provided with a material, a shape, and a size that do not affect the measurement of the ion current. 
     The materials of the individual electrode  112  and the common electrode  105  can be materials capable of performing an electron transfer reaction (Faraday reaction) with the electrolyte in the droplet  110  and the electrolyte solution  103 , and examples thereof include silver halide and alkali silver halide. Particularly, silver or silver/silver chloride can be used from the viewpoint of potential stability and reliability. 
     The materials of the individual electrode  112  and the common electrode  105  may be materials serving as a polarization electrode, and for example, gold or platinum can be used. In this case, in order to secure a stable ion current, for example, a substance capable of assisting the electron transfer reaction, such as potassium ferricyanide or potassium ferrocyanide, can be added to the measurement solution. Alternatively, for example, a substance capable of performing an electron transfer reaction such as ferrocenes can be immobilized on a surface of the polarization electrode. 
     All of the individual electrodes  112  and the common electrode  105  may be made of the above material, or a surface of a base material (copper, aluminum, or the like) may be covered with the above material. Shapes of the individual electrode  112  and the common electrode  105  are not particularly limited, and can be shapes in which a surface area coming into contact with the measurement solution is increased. The individual electrodes  112  and the common electrode  105  are bonded to the wirings, and an electrical signal is sent to a measurement circuit. 
     The water-repellent liquid  111  is a liquid that has insulating properties and phase-separates from water, and can have high affinity with the biopolymers in some cases. Examples of the water-repellent liquid  111  include silicone oil, fluorine-based oil, mineral oil, and the like. Such liquids are often used in techniques such as PCR and digital PCR. Since the water-repellent liquid  111  is used to convey the droplets  110  by EWOD, a liquid having low viscosity and high fluidity can be used as the water-repellent liquid  111 . 
     Although not illustrated, each of the first liquid tank  104 A and the second liquid tank  104 B has an injection port for injecting a liquid into the inside and a discharge port for discharging a liquid in the inside. 
     &lt;Method for Forming Nanopores&gt; 
       FIG. 3B  is a schematic diagram illustrating the biopolymer analysis device  300  in a state in which the nanopores  101  are formed in the thin film  102 . In the configuration of  FIG. 3A , since the nanopores  101  are not provided, the biopolymers cannot be analyzed. Thus, the nanopores  101  can be formed by applying a voltage value equal to or higher than a dielectric breakdown voltage of the thin film  102  between the plurality of individual electrodes  112  and the common electrode  105 . 
     The method for forming the nanopores  101  on the thin film  102  is not particularly limited, and for example, electron beam irradiation by a transmission electron microscope or the like, dielectric breakdown by voltage application, or the like can be used. For example, the method described in “Itaru Yanagi et al., Sci. Rep. 4, 5000 (2014)” can be used as the method for forming the nanopores  101 . 
     The formation of the nanopores  101  by the voltage application when the thin film  102  is made of Si 3 N 4  can be performed in the following procedure, for example. First, the thin film  102  made of Si 3 N 4  is hydrophilized by Ar/O 2  plasma (manufactured by Samco Inc.) under the conditions of 10 WW, 20 sccm, 20 Pa, and 45 sec. Subsequently, the biopolymer analysis device  300  including the thin film  102  is set in a flow cell. Thereafter, the individual electrodes  112  and the common electrode  105  are introduced into each of the first liquid tank  104 A and the second liquid tank  104 B. Then, the droplet  110 , which is an electrolyte solution of pH 7.5 containing 1 M of CaCl 2 ) and 1 mM of Tris-10 mM of EDTA, is conveyed to the first liquid tank  104 A, and the second liquid tank  104 B is filled with the electrolyte solution. 
     The voltage is applied not only when the nanopores  101  are formed, but also when the ion current flowing through the nanopores  101  after the nanopores  101  are formed is measured. Here, the first liquid tank  104 A positioned on a GND electrode side is referred to as a cis tank, and the second liquid tank  104 B positioned on a variable voltage side is referred to as a trans tank. A voltage V cis  applied to an electrode on the cis tank side is set to 0 V, and a voltage V trans  is applied to an electrode on the trans tank side. The voltage V trans  is generated by, for example, a pulse generator (41501B SMU AND Pulse Generator Expander, manufactured by Agilent Technologies, Inc.). 
     A current value after pulse application can be read by an ammeter  106  (4156B PRECISION SEMICONDUCTOR ANALYZER, manufactured by Agilent Technologies, Inc.). A process of applying a voltage in order to form the nanopores  101  and a process of reading the ion current value are controlled by, for example, a self-written program (Excel VBA, Visual Basic for Applications) stored in the storage of the computer  108 . A current value condition (threshold current) is selected according to a diameter of the nanopore  101  formed before application of a pulse voltage, and a target diameter is obtained while the diameter of the nanopore  101  is sequentially increased. 
     The diameter of the nanopore  101  can be estimated from the ion current value. A criterion for the condition selection is as represented in Table 1, for example, when the material of the thin film  102  is Si 3 N 4  and the thickness of the thin film  102  is 5 nm. Here, an n-th pulse voltage application time t n  (where, n&gt;2.) is decided by the following Equation. 
         t   n =10 −3+(1/6)(n−1) −10 −3+(1/6)(n−2)  For n&gt;2  [Equation 1]
 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Voltage application condition 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Nanopore diameter 
                 Non-opening  
                 φ1.4 nm 
               
               
                   
                 before pulse voltage 
                 to φ0.7 nm 
                   
               
               
                   
                 application 
                   
                   
               
               
                   
                 Applied voltage  
                 5 
                 3.5 
               
               
                   
                 (V trans ) [V] 
                   
                   
               
               
                   
                 Initial application  
                 0.001 
                 0.01 
               
               
                   
                 time [s] 
                   
                   
               
               
                   
                 Threshold current 
                 0.1 nA/0.4 V 
                 0.4 nA/0.1 V 
               
               
                   
                   
               
            
           
         
       
     
     The nanopores  101  can be formed not only by the method for applying the pulse voltage but also by electron beam irradiation by TEM (A. J. Storm et al., Nat. Mat. 2 (2003)). 
     A dimension of the nanopore  101  can be selected according to a type of the biopolymer to be analyzed. The dimension thereof can be, for example, 0.9 nm to 100 nm, and can be 0.9 nm to 50 nm in some cases. Specifically, the dimension of the nanopore  101  is equal to or more than 0.9 nm and equal to or less than 10 nm. For example, the diameter of the nanopore  101  used for analyzing single-stranded DNA having a diameter of about 1.4 nm can be, for example, 0.8 nm to 10 nm or 0.8 nm to 1.6 nm. For example, the diameter of the nanopore  101  used for analyzing double-stranded DNA having a diameter of about 2.6 nm can be 3 nm to 10 nm or 3 nm to 5 nm. 
     A depth of the nanopore  101  can be adjusted by adjusting the thickness of the thin film  102 . The depth of the nanopore  101  can be two times or more a monomer unit constituting the biopolymer, and can be three times or more or five times or more in some cases. For example, when the biopolymer is a nucleic acid, the depth of the nanopore  101  is three or more bases, for example, about 1 nm or more. In this manner, the biopolymers can enter the nanopores  101  while a shape and a moving speed thereof are controlled, and highly sensitive and highly accurate analysis can be performed. The shape of the nanopore  101  is basically circular, and may be elliptical or polygonal. 
     Immediately before a user analyzes the biopolymers by using the biopolymer analysis device  300 , in a state in which the droplets  110  are conveyed to the positions coming into contact with the individual electrodes  112  and are insulated from each other by the water-repellent liquid  111  as illustrated in  FIG. 3B , the nanopores  101  are provided in the thin film  102  by the electrical operation, and thus, it is possible to constantly provide the nanopores  101  with high quality. 
     The biopolymer analysis device  300  may be provided to the user in a state in which the droplets  110  and the water-repellent liquid  111  are conveyed to the positions illustrated in  FIG. 3A . Alternatively, the biopolymer analysis device may be provided to the user in a state in which only the water-repellent liquid  111  is introduced into the first liquid tank  104 A, and the droplets  110  may be conveyed to the positions illustrated in  FIG. 3A  by applying the EWOD conveying voltage to the individual electrodes  112  by an operation of the user. The biopolymer analysis device  300  may be provided to the user in a state in which both the first liquid tank  104 A and the second liquid tank  104 B are empty. In this case, the biopolymer analysis device is in the state illustrated in  FIG. 3A  by filling the first liquid tank  104 A with the water-repellent liquid  111  by an operation of the user, conveying the droplets  110  by the application of the EWOD conveying voltage to the individual electrodes  112 , and introducing the electrolyte solution  103  into the second liquid tank  104 B. 
     &lt;Another Configuration Example of Biopolymer Analysis Device&gt; 
       FIG. 4  is a schematic diagram illustrating another biopolymer analysis device  400  according to the first embodiment. The biopolymer analysis device  400  has a configuration adopting the configuration of the present embodiment ( FIG. 3 ) for a typical solid-state nanopore device used for analyzing the biopolymers by a blockade current method. As illustrated in  FIG. 4 , the biopolymer analysis device  400  includes a thin film  102 A made of an inorganic material, a tapered layer  102 B disposed on one side of the thin film  102 A, and a sacrificial layer  102 C disposed on the other side of the thin film  102 A. The thin film  102 A, the tapered layer  102 B, and the sacrificial layer  102 C may be collectively referred to as a “thin film”. 
     Silicon (Si) is generally adopted as the materials of the tapered layer  102 B and the sacrificial layer  102 C in consideration of mass productivity. The tapered layer  102 B is formed by, for example, anisotropic etching of a silicon wafer. The sacrificial layer  102 C has a plurality of (three in  FIG. 4 ) etching holes (protrusions) formed by, for example, etching of a silicon wafer at positions facing the plurality of individual electrodes  112 , and thus, the thin film  102 A is exposed at a plurality of portions to achieve an array. The sacrificial layer  102 C supports the thin film  102 A by stress. The configuration of such a solid-state nanopore device is described, for example, in U.S. Pat. No. 5,795,782, “Yanagi, et al., Scientific Reports 4, 5000, 2014”, “Akahori, et al., Nanotechnology 25 (27): 275501, 2014”, and “Yanagi, et al., Scientific Reports, 5, 14656, 2015”. 
     A dimension of the thin film  102 A exposed to the droplets  110  needs to be an area in which it is difficult to form two or more nanopores  101  when the nanopores  101  are formed by the application of the voltage, and an area allowable in terms of strength. The area is, for example, about 100 to 500 nm, and a film thickness at which the nanopores  101  having an effective film thickness equivalent to a single base can be formed is appropriately about 3 to 7 nm in order to achieve DNA single base resolution. 
     As illustrated in  FIG. 4 , in the case of a configuration in which a plurality of individual solution tanks is arrayed, the exposed portions of the thin film  102  where the nanopores  101  are formed can be regularly sequenced. An interval between the plurality of exposed portions of the thin film  102 A can be set to, for example, 0.1 mm to 10 mm or 0.5 mm to 4 mm according to the capability of an electrode and an electric measurement system to be used. 
       FIG. 5  is a schematic diagram illustrating another biopolymer analysis device  500  according to the first embodiment. As illustrated in  FIG. 5 , the biopolymer analysis device  500  is different from the biopolymer analysis device  400  illustrated in  FIG. 4  in that a plurality of tapered layers  102 B is provided. Such a configuration is described in, for example, “Yanagi, et al., Lab on a Chip, 16, 3340-3350, 2016.”. 
     &lt;Biopolymer Analysis Method&gt; 
     Hereinafter, a method for continuously performing the formation of the nanopores and the analysis of the biopolymers by using the biopolymer analysis device before the formation of the nanopores will be described. In a biopolymer analysis method according to the present embodiment, any one of the biopolymer analysis devices  300  to  500  illustrated in  FIGS. 3A, 4, and 5  may be used, and the common electrode  105  and the plurality of individual electrodes  112  are connected to the ammeter  106 , the power supply  107 , and the computer  108  illustrated in  FIGS. 1 and 2 . The biopolymer analysis device in which the first liquid tank  104 A and the second liquid tank  104 B are empty is used. 
       FIG. 6  is a flowchart illustrating the biopolymer analysis method using the biopolymer analysis device according to the present embodiment. First, in step S 1 , the user introduces the water-repellent liquid  111  from the injection port (not illustrated) of the first liquid tank  104 A (individual electrode  112  side), and fills the first liquid tank  104 A with the water-repellent liquid  111 . 
     In step S 2 , the user inputs an operation start instruction to the computer  108 , and sequentially injects the plurality of droplets  110  into the injection port (not illustrated) of the first liquid tank  104 A. Here, the plurality of droplets  110  is electrolyte solutions for opening the nanopores. 
     When the operation start instruction is received, the computer  108  applies the EWOD conveying voltage to the individual electrode  112  by the power supply  107 , and conveys the plurality of droplets  110  such that each droplet  110  is disposed at the position in contact with one individual electrode  112 . The water-repellent liquid  111  prevents the droplets  110  from coming into contact with each other and electrically insulates the droplets  110  from each other. Accordingly, the plurality of independent individual solution tanks (the plurality of channels) each having one individual electrode  112  and one droplet  110  is formed. 
     In step S 3 , the computer  108  detects the positions where the plurality of droplets  110  is conveyed. Subsequently, in step S 4 , the computer  108  determines whether or not the droplets  110  are moved to desired positions. A method for determining the positions of the droplets  110  will be described later. When the droplet  110  does not reach the desired positions (No), the processing returns to step S 2 , and the computer  108  repeats the conveyance of the droplets  110  until the droplets reach the desired position. 
     After the droplets  110  reach the desired positions (Yes in step S 4 ), in step S 5 , the computer  108  applies a voltage for reading a leakage current between the individual electrodes  112  of the adjacent channels, and measures a leakage current value. 
     In step S 6 , the computer  108  determines whether or not the measured leakage current value is less than a preset threshold. 
     When the leakage current value is equal to or more than the threshold (No in step S 6 ), since the channel does not maintain electrical independence, the processing returns to step S 2 , and the computer  108  tries again from the conveyance of the droplets  110  to the measurement of the leakage current until the leakage current value becomes less than the threshold. Alternatively, instead of returning to step S 2 , the computer  108  determines that the channel is defective and abandons the use of the channel. At this time, the computer  108  stores the position of the channel determined to be defective in the storage. 
     When the leakage current value is less than the threshold (Yes in step S 6 ), it can be determined that the channel is favorable, and thus, the processing proceeds to step S 7 . 
     After the droplets  110  are moved to all the channels and the electrical independence is confirmed, in step S 7 , the user introduces the electrolyte solution  103  into the second liquid tank  104 B. 
     In step S 8 , the computer  108  electrically opens the nanopores  101  by applying a voltage equal to or more than the dielectric breakdown voltage of the thin film  102  between each individual electrode  112  and the common electrode  105 . The computer  108  measures current-voltage characteristics of the nanopores  101  by applying a voltage for determining nanopore characteristics between each of the independent individual electrodes  112  and the common electrode  105 . Here, when the measured current value falls within a desired current value range, that is, within a desired nanopore diameter range, it is determined that the favorable nanopores  101  are obtained. 
     When the measured current value is out of the desired range, the computer  108  determines that the channel is a defective portion, and abandons the use of the channel. In this case, the computer  108  stores positional information of the abandoned channel in the storage so as not to move the droplet containing a sample to the abandoned channel. Accordingly, it is possible to prevent a loss of the sample. 
     Since the droplet  110  conveyed to the individual electrode side by the above operation is the electrolyte solution for opening the nanopores, it is necessary to replace the electrolyte solution with a solution for measuring the sample. In step S 9 , the computer  108  applies the EWOD conveying voltage to the individual electrode  112 , conveys the droplets  110 , which are nanopore opening solutions, to the discharge port of the first liquid tank  104 A, and moves the droplets to a waste liquid tank (not illustrated) connected to the discharge port. 
     Thereafter, the user injects the droplets (sample solutions) for measuring the sample containing the biopolymers from the injection port of the first liquid tank  104 A, and the computer  108  moves the sample solution to a portion where the favorable nanopores  101  are formed by applying the EWOD conveying voltage to the individual electrode  112 . 
     After all the sample solutions are conveyed, in step S 10 , the computer  108  measures the sample by applying the sample measuring voltage between each individual electrode  112  and the common electrode  105 . 
     When the sample is replaced, an operation similar to that in step S 9  is executed. Specifically, the computer  108  applies the EWOD conveying voltage to the individual electrode  112 , conveys the sample solution for which measurement is completed to the discharge port of the first liquid tank  104 A, and moves the sample solution to the waste liquid tank connected to the discharge port. Thereafter, the user introduces a new sample solution from the injection port of the first liquid tank  104 A, and the computer  108  conveys the new sample solution by applying the EWOD conveying voltage to the individual electrode  112 . As described above, the solution in each individual solution tank can be smoothly replaced by the EWOD. 
     &lt;Method for Determining Position of Droplet&gt; 
     Next, a method for detecting the positions of the droplets  110  in steps S 3  and S 4  described above will be described. Whether or not the droplets  110  reach the desired positions can be detected by various methods. For example, a transparent substrate and transparent electrodes are used as the substrate  113  and the individual electrodes  112 , and an observation device such as a microscope (a mechanism for determining whether or not the plurality of droplets is conveyed to the desired positions) is provided above the individual electrodes  112  and the substrate  113 . In this manner, it is possible to optically observe the images of inside of the first liquid tank  104 A. The observation device is configured to be able to transmit image data obtained by imaging an observation portion to the computer  108 . The computer  108  can determine the positions of the droplets  110  based on the image data. 
     On the other hand, when opaque materials are used for the individual electrodes  112  and the substrates  113 , the images of the droplets  110  cannot be observed. In this case, the positions of the droplets  110  can be determined by using an electrical method instead of the optical method described above. Since the droplets  110  conveyed by the biopolymer analysis device according to the present embodiment contain the electrolyte, the droplets are electrically conducted. Thus, it is possible to determine whether or not the droplets  110  come into contact with the individual electrodes  112  (the droplets  110  are at the positions of the individual electrodes  112 ) by applying an electrical operation between the individual electrodes  112  or between each individual electrode  112  and the common electrode  105  and examining whether or not an electrical reaction changes. 
     For example, impedance characteristics at the time of AC application vary depending on whether the individual electrodes  112  come into contact with the water-repellent liquid  111  containing the electrolyte or the electrolyte solution. Accordingly, it can be determined whether or not the droplets  110  come into contact with the individual electrode  112  by applying the alternating current to the individual electrodes  112  and measuring the impedance. 
     Alternatively, the positions of the droplets  110  can also be determined by measuring the current value between the individual electrode  112  and the common electrode  105  and examining resistance characteristics. For example, when the water-repellent liquid  111  comes into contact with the individual electrodes  112  and the thin film  102 , since the individual electrodes  112  and the common electrode  105  are completely insulated from each other by high insulating properties of the water-repellent liquid  111 , the observed current value is 10 −13  to 10 −14  Å or less. On the other hand, in a state in which the electrolyte solutions such as the droplets  110  come into contact with the individual electrodes  112  and the thin film  102 , since the electrolyte solution is a low resistor, a current value of 10 −11  to 10 −12  Å is observed between the individual electrodes  112  and the common electrode  105  even before the nanopores  101  are opened. A case where such a current value is observed is reported in, for example, “Scientific Reports, 5, 14656, 2015, Yanagi, et al.”. As described above, it is possible to determine whether the droplets  110  come into contact with the individual electrodes  112  and the thin film  102  by detecting a difference between the current values, and thus, it is possible to determine the positions of the droplets  110 . 
     Technical Effects 
     As described above, in the first embodiment, the plurality of droplets  110  is automatically moved to the desired positions by applying the EWOD conveying voltage to the individual electrode  112 , and thus, it is possible to collectively inject the solutions into the plurality of independent individual solution tanks. At this time, the droplets  110  are electrically insulated from each other by the presence of the water-repellent liquid  111 , and the electrical independence is maintained. When the solution is replaced, since the droplets  110  are conveyed by the EWOD and abandoned and new droplets  110  are similarly conveyed to desired positions, the solution can be smoothly replaced. Accordingly, it is possible to achieve both the collective injection of the solutions into the plurality of independent individual solution tanks and the solution replacement of the individual solution tanks while maintaining the insulation between the parallel channels. Since a liquid feeding device for conveying or replacing the solution is unnecessary, an increase in size of the device and an increase in installation cost can be avoided. 
     The EWOD exhibits an effect even when a degree of integration is high, that is, when a component dimension is minute. In particular, since the EWOD can convey even microdroplets of several μL to several nL, it is possible to measure the sample with the small amount of droplets. 
     In the biopolymer analysis device according to the present embodiment, the independent individual solution tanks can be integrated. Accordingly, it is possible to simultaneously measure different types of samples. For example, a certain droplet as a solution of a sample A and another droplet as a solution of a sample B are prepared, and the droplets are conveyed to appropriate positions. Thus, samples of different types can be simultaneously measured. When the biopolymer analysis device according to the present embodiment is used as, for example, a DNA sequencer, the sample A having a genetic mutation A and the sample B having a genetic mutation B can be separately and simultaneously measured on one device. The same applies to a gene detection method based on hybridization with a probe fixed. Alternatively, DNA sequencing and the above-described hybridization detection method or the like can be performed simultaneously. As described above, the throughput of the measurement can be improved by integrating the individual solution tanks. 
     Second Embodiment 
     In general, when the droplets are conveyed by the EWOD, an insulator (dielectric) may be installed on the electrode surface in order to enhance wettability to the electrode surface by drawing and polarizing electric charges from the surface of the droplet. However, when the insulator is installed on the surface of the individual electrode  112  of the first embodiment, it is difficult to measure the current due to high insulation resistance, and the biopolymers cannot be analyzed by using the individual electrode  112 . 
     In order to solve such a problem, in a second embodiment, as individual electrodes, two types of electrodes of one or more electrodes for current measurement and electrodes for EWOD are separately installed for the droplets. 
     &lt;Configuration Example of Biopolymer Analysis Device&gt; 
       FIG. 7  is a schematic diagram illustrating a biopolymer analysis device  700  according to the second embodiment. The biopolymer analysis device  700  is different from the biopolymer analysis device  400  illustrated in  FIG. 4  in the configuration of a substrate  113 . Accordingly, configurations other than the substrate  113  are not described. 
     As illustrated in  FIG. 7 , a plurality of individual electrodes  112  (a plurality of third electrodes) for current measurement and a plurality of EWOD electrodes  114  (a plurality of first electrodes) are embedded in the substrate  113 . The plurality of individual electrodes  112  is arranged at positions facing exposed portions of the thin film  102 A. Insulators  115  are provided on inner surfaces of the EWOD electrodes  114 . As will be described later, the plurality of EWOD electrodes  114  is arranged so as to form lanes for conveying droplets  110  at positions coming into contact with the individual electrodes  112 . 
       FIG. 7  illustrates a state in which the droplets  110  are conveyed to desired positions, and each droplet  110  comes into contact with at least one individual electrode  112  and the plurality of EWOD electrodes  114  surrounding the individual electrode. In this manner, EWOD conveyance and current measurement can be performed without problems by providing the electrodes for current measurement and the EWOD electrodes as separate applications. 
     &lt;Biopolymer Analysis Method&gt; 
     The biopolymer analysis method according to the present embodiment is substantially the same as that of the first embodiment ( FIG. 6 ), but is different from that of the first embodiment in that an EWOD conveying voltage is applied to the EWOD electrodes  114  instead of the individual electrodes  112  in the conveyance of the droplets in steps S 2  and S 9 . 
       FIG. 8A  is a top view of the biopolymer analysis device  700 . As illustrated in  FIG. 8A , on the substrate  113 , a total of 16 individual electrodes  112  for current measurement of 4 columns×4 rows are arranged, and the plurality of EWOD electrodes  114  is arranged around each individual electrode  112 . Thus, the plurality of EWOD electrodes  114  form a lane for conveying the droplet  110 , and can smoothly convey the droplet  110 . Each individual electrode  112  is disposed above the exposed portion of the thin film  102 . When each individual electrode  112  is a transparent electrode, as illustrated in  FIG. 8A , the thin film  102  can be observed from above the individual electrode  112 . The state illustrated in  FIG. 8A  is a state after the water-repellent liquid  111  is introduced in step S 1  ( FIG. 6 ) described in the first embodiment. 
       FIGS. 8B and 8C  are top views of the biopolymer analysis device  700  illustrating a scene in which the droplet  110  is conveyed. As described above, the droplet  110  is conveyed by applying the EWOD conveying voltage to the EWOD electrodes  114 . As illustrated in  FIG. 8B , for example, when the droplets  110  conveyed via a channel of a flow cell are introduced into the first liquid tank  104 A and come into contact with the EWOD electrodes  114  to which the EWOD conveying voltage is applied, the droplets  110  can be transported discretely by one electrode. Finally, one droplet  110  is disposed between the thin film  102  and the individual electrode  112 . As illustrated in  FIG. 8C , the droplets  110  can be arranged between all the exposed portions of the thin film  102  and the individual electrodes  112  by similarly repeating this operation. 
     The numbers and arrangement layouts of the individual electrodes  112  and the EWOD electrodes  114  are not limited to those illustrated in  FIGS. 8A to 8C , and can be appropriately changed. For example, when the channels are highly integrated, the individual electrodes  112  may be provided in units of several hundreds to several thousands or more. 
     Technical Effects 
     As described above, in the present embodiment, the configuration in which the individual electrode  112  for current measurement and the EWOD electrodes  114  are provided in the first liquid tank  104 A is adopted. Accordingly, even though the insulators  115  are provided on surfaces of the EWOD electrodes  114 , the formation of the nanopores and the measurement of the current can be performed without any problem by using the individual electrodes  112 . 
     Third Embodiment 
     As described above, when the insulators (dielectrics) are installed on the surfaces of the individual electrodes  112  of the first embodiment, it is difficult to measure the current due to the high insulation resistance, and the biopolymers cannot be analyzed by using the individual electrodes  112 . 
     In order to solve such a problem, in a third embodiment, a circuit for EWOD conveyance, a circuit for nanopore opening, and a circuit for current measurement are connected to each individual electrode  112 , and a voltage applied to the individual electrode  112  is controlled by switching between these circuits. 
     &lt;Configuration Example of Biopolymer Analysis Device&gt; 
       FIG. 9  is a schematic diagram illustrating a biopolymer analysis device  800  according to the third embodiment. A configuration of the biopolymer analysis device  800  is substantially the same as that of the biopolymer analysis device  400  of  FIG. 4  described in the first embodiment, but a control circuit  121  (controller) is connected to the individual electrodes  112  (the plurality of first electrodes) through wirings. As illustrated in  FIG. 9 , in the control circuit  121 , an EWOD conveying circuit  116 , a nanopore opening circuit  117 , a current measuring circuit  118 , and a plurality of switches  122  for switching between these circuits are provided. The control circuit  121  is connected to the computer  108  (controller). The switching of the switches  122  and the application of the voltage using the circuits  116  to  118  are controlled by the computer  108 . 
     For example, a circuit having a configuration such as a capacitor  123  (insulator) that appropriately draws electric charges from the droplets between the EWOD conveying circuit  116  and the individual electrodes  112  is provided, and thus, the EWOD conveyance can be appropriately performed without installing the insulators on the surfaces of the individual electrodes  112 . One EWOD conveying circuit  116  common to all the individual electrodes  112  may be provided. 
     &lt;Biopolymer Analysis Method&gt; 
     The biopolymer analysis method according to the present embodiment is substantially the same as that of the first embodiment ( FIG. 6 ), but is different from that of the first embodiment in that the computer  108  changes the voltage applied to the individual electrode  112  by switching between the switches  122 . Accordingly, only differences from the first embodiment will be described. 
     In step S 2 , the computer  108  connects the EWOD conveying circuit  116  and each individual electrode  112  by switching between the switches  122 , and applies the EWOD conveying voltage to each individual electrode  112 . 
     In step S 5 , the computer  108  connects the current measuring circuit  118  and each individual electrode  112  by switching between the switch  122 , applies a voltage for reading the leakage current between the individual electrodes  112  of the adjacent channels, and measures a leakage current value. 
     In step S 8 , the computer  108  connects the nanopore opening circuit  117  and each individual electrode  112  by switching between the switches  122 , applies a voltage equal to or more than the dielectric breakdown voltage of the thin film  102  between each individual electrode  112  and the common electrode  105 , and electrically opens the nanopores  101 . 
     In step S 9 , the computer  108  connects the EWOD conveying circuit  116  and each individual electrode  112  by switching between the switches  122 . Subsequently, the EWOD conveying voltage is applied to the individual electrode  112 , the droplets  110 , which are the nanopore opening solutions, are conveyed to the discharge port of the first liquid tank  104 A, and the droplets are moved to a waste liquid tank (not illustrated) connected to the discharge port. 
     In step S 10 , the computer  108  connects the current measuring circuit  118  and each individual electrode  112  by switching between the switches  122 , applies the sample measuring voltage between each individual electrode  112  and the common electrode  105 , and measures the sample. 
     Technical Effects 
     As described above, in the present embodiment, the configuration in which the EWOD conveying circuit  116 , the nanopore opening circuit  117 , and the current measuring circuit  118  are connected to the plurality of individual electrodes  112 , and the circuits connected to the individual electrodes  112  are switched by the switches  122  is adopted. Accordingly, it is possible to convey the droplets  110 , form the nanopores, and measure the current value only with the individual electrodes  112  and the common electrode  105  without separately providing the EWOD electrode. Therefore, it is possible to increase the number of channels per unit area of the biopolymer analysis device as compared with the second embodiment. 
     Fourth Embodiment 
     As illustrated in  FIGS. 4 and 5 , the solid-state nanopore device often has a structure in which the sacrificial layer  102 C that is a flat surface is provided on one side of the thin film  102 A and the tapered layer  102 B that is a tapered surface is provided on the other side. However, the sacrificial layer  102 C has a structure (etching hole) in which only a specific region is removed by chemical etching or dry etching in order to expose the thin film  102 A. 
     In some structures of the biopolymer analysis device, the water-repellent liquid  111  remains in the etching hole, and the droplet  110  cannot enter the etching hole. Thus, a problem of a defective channel is caused. 
       FIG. 10A  is a schematic diagram illustrating a state in which the water-repellent liquid  111  remains in an etching hole  102 D of the sacrificial layer  102 C. As illustrated in  FIG. 10A , when the etching hole  102 D has, for example, a cylindrical shape, the water-repellent liquid  111  enters first, and this space is a hydrodynamically immovable region. Thus, when the droplet  110  is conveyed onto the etching hole  102 D, the replacement is not promptly preformed fluidly, and the water-repellent liquid  111  remains in the etching hole  102 D. Such a phenomenon easily occurs in the water-repellent liquid often used in the EWOD. That is, since the water-repellent liquid has chemical properties such as low viscosity and low surface tension, a phenomenon in which the replacement is not performed in the structure having the immovable region like the cylindrical etching hole  102 D occurs. In particular, when the density of the water-repellent liquid  111  is higher than the density of the droplet, buoyancy acts reversely to the replacement, and thus, the replacement becomes more difficult. 
     A configuration for preventing the water-repellent liquid  111  from remaining in the etching hole  102 D of the sacrificial layer  102 C will be described below. 
       FIG. 10B  is a schematic diagram illustrating a structure of the sacrificial layer  102 C of the present embodiment. As illustrated in  FIG. 10B , in the sacrificial layer  102 C of the present embodiment, a cross-sectional shape of the etching hole  102 D (recess) is formed in a tapered shape. In this manner, the cross-sectional shape of the etching hole  102 D is formed so as not to have the fluidly immovable region such as the tapered shape, and thus, the water-repellent liquid  111  can be easily replaced fluidly by the droplet as the electrolyte solution. 
     When the etching hole  102 D has the cylindrical shape, the electrolyte solution is filled in the cylindrical etching hole  102 D in advance before the first liquid tank  104 A is filled with the water-repellent liquid  111 , and thus, the water-repellent liquid  111  can be prevented from remaining. Since the liquid in the cylindrical etching hole  102 D is less likely to be replaced fluidly, the water-repellent liquid  111  does not enter the etching hole  102 D when the water-repellent liquid  111  subsequently moves. In this case, the water-repellent liquid  111  is less likely to enter the etching hole  102 D by using a fluid having a specific gravity lower than that of water as the water-repellent liquid  111 . 
       FIG. 10C  is a schematic diagram illustrating another biopolymer analysis device  900  according to the present embodiment. As illustrated in  FIG. 10C , structures of a thin film  102 A, a tapered layer  102 B, and a sacrificial layer  102 C of the biopolymer analysis device  900  are similar to those of the biopolymer analysis device  500  of the first embodiment ( FIG. 5 ), but a substrate  113  on which the plurality of individual electrodes  112  is provided is disposed in a second liquid tank  104 B, and a common electrode  105  is disposed in a first liquid tank  104 A. A plurality of droplets  110  and a water-repellent liquid  111  are introduced into the second liquid tank  104 B, and an electrolyte solution  103  is introduced into the first liquid tank  104 A. 
     In this manner, the water-repellent liquid  111  can be fluidly and easily replaced with the droplets  110  by filling the tapered layer  102 B side (second liquid tank  104 B) with the water-repellent liquid  111  and then conveying the droplets  110 . 
     Technical Effects 
     As described above, in the present embodiment, the configuration in which the cross-sectional shape of the etching hole  102 D formed in the sacrificial layer  102 C is the tapered shape is adopted. Alternatively, the configuration in which the cylindrical etching hole  102 D is filled with the electrolyte solution in advance is adopted. It is also possible to adopt the configuration in which the plurality of individual electrodes  112  is provided on the tapered layer  102 B side (second liquid tank  104 B) and the water-repellent liquid  111  and the droplets  110  are introduced into the tapered layer  102 B side. In this manner, it is possible to prevent the water-repellent liquid  111  from remaining in the etching hole  102 D formed in the sacrificial layer  102 C and from being the defective channel. 
     Fifth Embodiment 
     &lt;Configuration Example of Biopolymer Analysis Device&gt; 
       FIG. 11  is a schematic diagram illustrating a biopolymer analysis device  1000  according to a fifth embodiment. As illustrated in  FIG. 11 , the biopolymer analysis device  1000  according to the present embodiment is different from the first embodiment ( FIG. 4 ) and the second embodiment ( FIG. 7 ) in that EWOD electrodes  114  are formed on an upper surface of a sacrificial layer  102 C (thin film). Insulators  115  are arranged on surfaces of the EWOD electrodes  114 . Each EWOD electrode  114  is connected to an external circuit through a wiring (not illustrated) provided inside the sacrificial layer  102 C. Droplets  110  are conveyed to positions coming into contact with one individual electrode  112  and coming into contact with at least two adjacent EWOD electrodes  114 . 
       FIG. 12  is a schematic diagram illustrating another biopolymer analysis device  1100  according to the fifth embodiment. As illustrated in  FIG. 11 , the biopolymer analysis device  1100  according to the present embodiment is different from the first embodiment ( FIG. 4 ) and the second embodiment ( FIG. 7 ) in that a plurality of individual electrodes  112  (a plurality of third electrodes) for current measurement is formed on an upper surface of a sacrificial layer  102 C (thin film), and only EWOD electrodes  114  (a plurality of first electrodes) are formed on a substrate  113 . Each individual electrode  112  is connected to an external circuit through a wiring (not illustrated) provided inside the sacrificial layer  102 C. Droplets  110  are conveyed to positions coming into contact with one individual electrode  112  and coming into contact with at least two adjacent EWOD electrodes  114 . In other words, each of the individual electrodes  112  is disposed so as to come into contact with one droplet  110 . 
     Technical Effects 
     As described above, each of the biopolymer analysis devices  1000  and  1100  according to the present embodiment includes the individual electrodes  112  for current measurement and the EWOD electrodes  114 , and adopt the configuration in which any one of the individual electrodes  112  or the EWOD electrodes  114  are integrated with the sacrificial layer  102 C on the thin film  102 A. Accordingly, as compared with the case where both the individual electrodes  112  for current measurement and the EWOD electrodes  114  are provided on the substrate  113  as in the second embodiment, the channels can be further integrated, and the measurement using the droplets having a smaller volume can be performed. 
     Sixth Embodiment 
     In the first embodiment, as illustrated in  FIG. 3A , the configuration in which the substrate  113  having the plurality of individual electrodes  112  is disposed on one side (first liquid tank  104 A) of the thin film  102  and the droplets  110  are introduced has been described. On the other hand, in a sixth embodiment, substrates  113  having a plurality of individual electrodes  112  are disposed on both sides (a first liquid tank  104 A and a second liquid tank  104 B) of a thin film  102 , and droplets  110  are introduced into both the first liquid tank  104 A and the second liquid tank  104 B. 
     &lt;Configuration Example of Biopolymer Analysis Device&gt; 
       FIG. 13  is a schematic diagram illustrating a biopolymer analysis device  1200  according to the sixth embodiment. As illustrated in  FIG. 13 , the biopolymer analysis device  1200  according to the present embodiment includes a thin film  102 , a first liquid tank  104 A, a second liquid tank  104 B, a substrate  113 A having a plurality of individual electrodes  112 A (a plurality of first electrodes), and a substrate  113 B having a plurality of individual electrodes  112 B (a plurality of second electrodes). The substrate  113 A is provided in the first liquid tank  104 A, and the substrate  113 B is provided in the second liquid tank  104 B. The plurality of individual electrodes  112 A and the plurality of individual electrodes  112 B are arranged at positions facing each other with the thin film  102  interposed therebetween. 
     A plurality of droplets  110  (measurement solutions) and a water-repellent liquid  111  are introduced into the first liquid tank  104 A and the second liquid tank  104 B, respectively. Each droplet  110  is electrically insulated from the adjacent droplet  110  by the water-repellent liquid  111 , and the droplets are independent of each other. The plurality of droplets  110  comes into contact with the individual electrode  112 , respectively, and thus, an electrical operation such as application of a voltage can be performed on each droplet  110 . Other configurations are similar as those of the biopolymer analysis device  300  ( FIG. 3 ) according to the first embodiment, and thus, the description thereof is omitted. 
     &lt;Biopolymer Analysis Method&gt; 
     Since a biopolymer analysis method according to the present embodiment is substantially the same as that of the first embodiment, the biopolymer analysis method according to the present embodiment will be described with reference to  FIG. 6 . Steps similar to those in the first embodiment will not be described. 
     First, steps S 1  to S 6  of the first embodiment are performed, and the plurality of individual solution tanks is formed by introducing the water-repellent liquid  111  and the droplets  110  into the first liquid tank  104 A. Thereafter, instead of step S 7 , the plurality of individual solution tanks is formed by introducing the water-repellent liquid  111  and the droplets  110  into the second liquid tank  104 B as in steps S 1  to S 6 . 
     Subsequently, in step S 8 , the computer  108  electrically opens the nanopores  101  by applying the voltage equal to or more than the dielectric breakdown voltage of the thin film  102  between the individual electrodes  112 A and the individual electrodes  112 B facing each other. 
     In steps S 9  and S 10 , the droplets  110  for opening the nanopores are abandoned from the first liquid tank  104 A by applying the EWOD conveying voltage to the individual electrodes  112 A, the sample is measured by introducing the sample solution, and then the droplets  110  for opening the nanopores are replaced with the sample solution by similarly applying the EWOD conveying voltage to the individual electrodes  112 B in the second liquid tank  104 B. Thereafter, the sample can be measured for the sample solution introduced into the second liquid tank  104 B by reversing the voltage applied between the individual electrodes  112 A and the individual electrodes  112 B facing each other. 
     Technical Effects 
     As described above, in the present embodiment, the configuration in which the substrates  113  having the plurality of individual electrodes  112  are provided in both the first liquid tank  104 A and the second liquid tank  104 B and the droplets  110  are conveyed by the EWOD is adopted. Accordingly, as compared with the first embodiment in which the sample solution is introduced into only one liquid tank (first liquid tank  104 A), the number of samples can be measured twice without performing the replacement of the sample solution. 
     Seventh Embodiment 
     In the first embodiment, the configuration in which the first liquid tank  104 A is one layer has been described. The inside of the first liquid tank  104 A may have a two-layer structure of a layer for conveying the droplets  110  and a layer for measuring the sample. 
     &lt;Configuration Example of Biopolymer Analysis Device&gt; 
       FIG. 14A  is a schematic diagram illustrating a biopolymer analysis device  1300  according to a seventh embodiment. As illustrated in  FIG. 14A , in the biopolymer analysis device  1300  according to the present embodiment, a substrate  113  forming an upper surface of a first liquid tank  104 A is disposed, a substrate  119  is disposed substantially parallel to the substrate  113  inside the first liquid tank  104 A, and the first liquid tank  104 A has a two-layer structure. A plurality of EWOD electrodes  114  (a plurality of first electrodes) is provided in the substrate  113 . Each of the plurality of EWOD electrodes  114  is covered with insulators  115 . A plurality of individual electrodes  112  (a plurality of third electrodes) and a plurality of openings  120  through which droplets  110  conveyed between the substrate  113  and the substrate  119  can pass are provided in the substrate  119 . 
     The droplets  110  are conveyed by filling the first liquid tank  104 A with a water-repellent liquid  111 , introducing a plurality of droplets  110  into an upper layer (between the substrate  113  and the substrate  119 ) of the first liquid tank  104 A, and applying an EWOD conveying voltage between the adjacent EWOD electrodes  114 . When the droplets  110  are conveyed to positions of the openings  120 , the droplets  110  move to a lower layer (between the substrate  119  and a thin film  102 ) via the openings  120 . The droplets  110  can move from the upper layer to the lower layer of the first liquid tank  104 A by using gravity, buoyancy, or a difference in surface tension of a substrate surface with respect to water. 
     A hydrophilization treatment may be performed on the substrate  119  on a wall surface of the opening  120 . Accordingly, this makes it easier to move the droplets  110  to the lower layer. 
       FIG. 14B  is a schematic diagram illustrating a state in which the plurality of droplets  110  is arranged in the lower layer of the first liquid tank  104 A. As illustrated in  FIG. 14B , each individual electrode  112  is disposed so as to come into contact with one droplet  110  when each droplet  110  passes through the opening  120  and moves to the lower layer. In this manner, the nanopores can be opened in the thin film  102  and the sample can be measured by forming an individual solution tank in which one individual electrode  112  comes into contact with one droplet  110  and applying a dielectric breakdown voltage or a current measuring voltage between the individual electrode  112  and the common electrode  105 . 
     Technical Effects 
     As described above, the biopolymer analysis device according to the present embodiment has a two-layer structure in which the substrate  113  having the plurality of EWOD electrodes  114  and the substrate  119  having the plurality of individual electrodes  112  are provided in the first liquid tank  104 A. Accordingly, the plurality of EWOD electrodes  114  and the plurality of individual electrodes  112  can be arranged at higher density on each of the substrates  113  and  119  as compared with the second embodiment that adopts the configuration in which both the plurality of EWOD electrodes  114  and the plurality of individual electrodes  112  are provided on the substrate  113 . 
     Eighth Embodiment 
     In the first embodiment to the seventh embodiment, the configuration of the biopolymer analysis device has been mainly described. Hereinafter, in the present embodiment, a biopolymer analysis equipment using the biopolymer analysis device will be described. Any one of the biopolymer analysis devices according to the first embodiment to the seventh embodiment may be used as the biopolymer analysis device included in the biopolymer analysis equipment. 
     &lt;Configuration Example of Biopolymer Analysis Equipment&gt; 
       FIG. 15  is a schematic diagram illustrating a configuration example of biopolymer analysis equipment  1800 . As an example, the biopolymer analysis equipment  1800  includes the biopolymer analysis device  700  according to the second embodiment (see  FIG. 7 ), a control circuit  121 , and a computer  108  (controller). 
     As illustrated in  FIG. 15 , a plurality of droplets  110  (sample solution) containing biopolymers  1  is conveyed to a first liquid tank  104 A. Nanopores are not formed in a thin film  102 A. An electrolyte solution  103  is introduced into a second liquid tank  104 B. In this manner, the nanopores can be formed in the thin film  102 A by using the droplets  110  containing the biopolymers  1 , and the biopolymers  1  can be analyzed as it is. In this case, since it is not necessary to replace a solution for opening the nanopores with a sample solution, a measurement time can be shortened. 
     Although not illustrated, an EWOD conveying circuit, a nanopore opening circuit, a current measuring circuit, and a switch for switching between these circuits are provided inside the control circuit  121 . Each individual electrode  112  and a common electrode  105  are connected to the nanopore opening circuit and the current measuring circuit via wirings. EWOD electrodes  114  are connected to the EWOD conveying circuit via wirings. 
     An ammeter that measures an ion current (blockade current) flowing between each individual electrode  112  and the common electrode  105  is provided in the current measuring circuit. The ammeter includes an amplifier that amplifies the current flowing between the individual electrode  112  and the common electrode  105 , and an analog-to-digital converter. The ammeter is connected to the computer  108 , and the analog-to-digital converter outputs, as a digital signal, a value of the detected ion current to the computer  108 . 
     The computer  108  is, for example, a terminal such as a personal computer, a smartphone, or a tablet, and includes a data processing unit that processes various kinds of data and a storage that stores an output value of the ammeter, data calculated by the data processing unit, and the like. The data processing unit counts the biopolymers  1  and acquires monomer sequence information of the biopolymers  1  based on a current value of the ion current (blockade current) output from the ammeter. The data processing unit determines whether or not leakage occurs at the positions of the droplets  110  or between the droplets  110  and whether or not the nanopores are formed in the thin film  102  based on electrical characteristics such as a measured current value. 
     The computer  108  controls switching of the control circuit  121  between switches and application of a voltage to the common electrode  105 , each individual electrode  112 , and each EWOD electrode  114 . 
     The control circuit  121  and the computer  108  may be integrated with the biopolymer analysis device instead of providing the control circuit  121  and the computer  108  separately from the biopolymer analysis device  700  as illustrated in  FIG. 15 . 
     &lt;Analysis of Biopolymer&gt; 
     In the state illustrated in  FIG. 15 , when a voltage for opening the nanopores is applied between each individual electrode  112  and the common electrode  105 , the nanopores are formed in the thin film  102 A. Thereafter, when a current measuring voltage is subsequently applied between the individual electrode  112  and the common electrode  105 , a potential difference is generated between both surfaces of the thin film  102 A, and the biopolymer  1  dissolved in the droplet  110  is migrated toward the common electrode  105 . When the biopolymer  1  is DNA, since the biopolymer is negatively charged in the droplet  110 , the biopolymer  1  can be migrated toward the common electrode  105  by using the common electrode  105  as a positive electrode. When the biopolymer  1  passes through the nanopore, a blockade current flows. 
     In the measurement of the blockade current using the biopolymer analysis device, a current value measured in the absence of the biopolymer  1  is used as a reference (pore current), a decrease in current observed when the nanopore surrounds the biopolymer  1  (blockade of the nanopore by the biopolymer  1 ) is measured, and a passage speed and state of a molecule are observed. When the biopolymer  1  finishes passing through the nanopore, the acquired current value returns to the pore current. From this blockade time, a nanopore passage speed of the biopolymer  1  can be analyzed, and characteristics of the biopolymer  1  can be analyzed from a blockade amount. 
     In the nanopore method for analyzing the biopolymer by the electrical signal, particularly a signal change of the ion current, as the electrical conductivity of the electrolyte solution is higher, a signal change amount of the ion current is larger. Thus, it is possible to perform measurement at a high SN ratio. Although depending on the transference number of ionic species and the like, generally, the electrical conductivity of the electrolyte solution can be increased by increasing ionic strength, that is, salt concentration. Accordingly, in the nanopore analysis, measurement is performed at a highest possible salt concentration from the viewpoint of the SN ratio. In particular, in the nanopore analysis, a potassium chloride aqueous solution having a concentration of 1 M is often used, and a high salt concentration condition having an ionic strength of 3 M or more is used in some cases. A maximum salt concentration is a saturated concentration that is an upper limit at which the electrolyte can be dissolved. 
     Specifically, for example, when the individual electrode  112  and the common electrode  105  are silver/silver chloride electrodes, a potassium chloride aqueous solution having a concentration of 3 M can be used as the droplets  110  and the electrolyte solution  103 . The reason is that since chloride ions can undergo an electron transfer reaction with the silver/silver chloride electrodes and potassium ions have the same electrical mobility as the chloride ions, the electrical conductivity can be sufficiently secured. In addition to potassium chloride, other kinds of monovalent cation of an alkali metal may be used as ionic species such as a lithium ion, a sodium ion, a rubidium ion, a cesium ion, or an ammonium ion. 
     &lt;Conveyance Control of Biopolymer&gt; 
     When DNA sequencing or RNA sequencing is performed by using the biopolymer analysis equipment  1800 , it is necessary to perform conveyance control when DNA or RNA passes through the nanopore. The conveyance control of the biopolymer can be mainly performed by a molecular motor using an enzyme. The conveyance control by the molecular motor needs to be started only in the vicinity of the nanopore. In particular, the start of the conveyance by the molecular motor in the vicinity of the nanopore can be controlled by binding a control chain to the biopolymer to be read. Such a configuration is described in, for example, JP application No. 2018-159481 and International application No. PCT/JP2018/039466. The disclosures of these documents are incorporated as constituting a part of the present specification. 
     Here, the enzyme used as the molecular motor refers to all enzymes having a binding capacity to the biopolymer. When the biopolymer is DNA, examples of the enzyme include DNA polymerase, DNA helicase, DNA exonuclease, DNA transposase, and the like. When the biopolymer is RNA, examples of the enzyme include RNA polymerase, RNA helicase, RNA exonuclease, RNA transposase, and the like. 
     As described above, when a voltage is applied to both ends of the nanopore disposed in the electrolyte solution, an electric field is generated in the vicinity of the nanopore  101 , and the biopolymer passes through the nanopore by the force. On the other hand, since the molecular motor is generally larger than a nanopore diameter, the molecular motor cannot pass through the nanopore. In order to realize this limitation, it is desirable that the nanopore diameter is in a range from 0.8 nm, which is a lower limit at which single-stranded DNA or single-stranded RNA can pass, to 3 nm, which is an upper limit at which the enzyme as the molecular motor does not pass. Under this condition, a primer in the control chain approaches the molecular motor staying in the vicinity of the nanopore, and thus, an extension and separation reaction is started. As a result, the biopolymer is pulled up or pulled down from the nanopore by the force when the molecular motor extends and separates a complementary chain, and the biopolymer is analyzed from a change in the ion current acquired at this time. 
     The configuration in which the monomer sequence information in the biopolymer  1  is acquired based on the electrical signal has been described above. The monomer sequence information of the biopolymer  1  can also be obtained by a method for acquiring a tunnel current by providing an electrode inside the nanopore or a method for detecting a change in transistor characteristics. The monomer sequence information of the biopolymer  1  may be acquired based on an optical signal. That is, a method for deciding each monomer sequence by providing a label having a characteristic fluorescence wavelength for each monomer and measuring the fluorescence signal may be used. 
     The biopolymer analysis device (nanopore device) for analyzing the biopolymer and the biopolymer analysis equipment including the same include the above-described components as elements. The biopolymer analysis device and the biopolymer analysis equipment can be provided together with instructions describing a use procedure, a use amount, and the like. The biopolymer analysis device may be provided in a state in which the nanopore is formed in an immediately usable state, or may be provided in a state in which the nanopore is formed in a providing destination. 
     Modification Example 
     The present disclosure is not limited to the above-described embodiments, and includes various modification examples. For example, the aforementioned embodiments are described in detail in order to facilitate easy understanding of the present disclosure, and are not limited to necessarily include all the described components. A part of a certain embodiment may be replaced with the configuration of another embodiment. The configuration of another embodiment can be added to the configuration of a certain embodiment. A part of the configuration of another embodiment can be added, deleted, or replaced to, from, or with a part of the configuration of each embodiment. 
     All publications and patent literatures cited in the present specification are hereby incorporated by reference in the present specification. 
     REFERENCE SIGNS LIST 
     
         
           1  biopolymer 
           101  nanopore 
           102  thin film 
           103  electrolyte solution 
           104 A first liquid tank 
           104 B second liquid tank 
           105  common electrode 
           106  ammeter 
           107  power supply 
           108  computer 
           110  droplet 
           111  water-repellent liquid 
           112  individual electrode 
           113  substrate 
           114  EWOD electrode 
           115  insulator 
           116  EWOD conveying circuit 
           117  nanopore opening circuit 
           118  current measuring circuit 
           119  substrate 
           120  opening 
           121  control circuit 
           122  switch 
           123  capacitor