阅读说明：本技术 生物体聚合物分析设备、生物体聚合物分析装置以及生物体聚合物分析方法 (Biopolymer analysis device, and biopolymer analysis method ) 是由 后藤佑介 藤冈满 中川树生 柳川善光 板桥直志 于 2019-04-24 设计创作，主要内容包括：本公开的生物体聚合物分析设备具备：绝缘性的薄膜,包含无机材质；第1液槽以及第2液槽,由所述薄膜隔开；多个第1电极,配置在所述第1液槽；以及第2电极,配置在所述第2液槽,在所述第1液槽导入疏水液以及多个液滴,所述多个第1电极构成为能够通过施加给定的电压,从而通过电介质上电润湿来输送导入到所述第1液槽的所述多个液滴,所述多个液滴被输送到与所述多个第1电极接触的部位,通过所述疏水液相互绝缘。(The disclosed biopolymer analysis device is provided with: an insulating film containing an inorganic material; a1 st liquid tank and a 2 nd liquid tank partitioned by the film; a plurality of 1 st electrodes disposed in the 1 st liquid bath; and a 2 nd electrode disposed in the 2 nd liquid tank, a hydrophobic liquid and a plurality of droplets are introduced into the 1 st liquid tank, the plurality of 1 st electrodes are configured to be capable of transferring the plurality of droplets introduced into the 1 st liquid tank by electrowetting on a dielectric by applying a predetermined voltage, the plurality of droplets are transferred to a portion in contact with the plurality of 1 st electrodes, and are insulated from each other by the hydrophobic liquid.)

1. A biopolymer analysis device is provided with:

an insulating film containing an inorganic material;

a1 st liquid tank and a 2 nd liquid tank partitioned by the film;

a plurality of 1 st electrodes disposed in the 1 st liquid bath; and

a 2 nd electrode disposed in the 2 nd liquid bath,

introducing hydrophobic liquid and a plurality of liquid drops into the first liquid tank 1,

the plurality of 1 st electrodes are configured to be capable of transporting the plurality of droplets introduced into the 1 st liquid tank by electrowetting on a dielectric by applying a predetermined voltage,

the plurality of droplets are transported to a portion in contact with the plurality of 1 st electrodes, and are insulated from each other by the hydrophobic liquid.

2. The biopolymer analysis device of claim 1, wherein,

the 1 st liquid tank is also provided with a plurality of 3 rd electrodes,

the plurality of droplets are respectively delivered to the parts contacting with the plurality of No. 1 electrodes and the plurality of No. 3 electrodes,

the plurality of No. 3 electrodes are configured to measure a current flowing from each of the plurality of droplets to the No. 2 liquid tank through the thin film.

3. The biopolymer analysis device of claim 1, wherein,

the plurality of 1 st electrodes have an insulating film on a surface thereof.

4. The biopolymer analysis device of claim 1, wherein,

the plurality of 1 st electrodes are also configured to be capable of measuring a current flowing from each of the plurality of droplets to the 2 nd liquid tank through the thin film.

5. The biopolymer analysis device of claim 2, wherein,

the thin film forms a nanopore by applying an insulation breakdown voltage of the thin film between the plurality of No. 3 electrodes and the No. 2 electrode.

6. The biopolymer analysis device of claim 4, wherein,

the thin film forms a nanopore by applying an insulation breakdown voltage of the thin film between the plurality of 1 st electrodes and the 2 nd electrode.

7. The biopolymer analysis device of claim 2, wherein,

the plurality of 1 st electrodes are arranged around the plurality of 3 rd electrodes, and form a passage for transporting the plurality of droplets.

8. The biopolymer analysis device of claim 1, wherein,

the biopolymer analysis device further includes a mechanism for determining whether or not the plurality of droplets are transported to a desired position.

9. The biopolymer analysis device of claim 1, wherein,

the thin film has a concave portion having a tapered cross-sectional shape at a portion where the droplet is transported.

10. The biopolymer analysis device of claim 2, wherein,

any one of the plurality of 1 st electrodes or the plurality of 3 rd electrodes is disposed on the thin film.

11. The biopolymer analysis device of claim 1, wherein,

a plurality of the 2 nd electrodes are arranged in the 2 nd liquid tank, the hydrophobic liquid and the plurality of liquid drops are introduced,

the plurality of 2 nd electrodes are configured to be capable of transporting the plurality of droplets introduced into the 2 nd liquid tank by electrowetting on a dielectric by applying the given voltage,

the plurality of droplets are transported to a portion in contact with the plurality of 2 nd electrodes, and are insulated from each other by the hydrophobic liquid.

12. A biopolymer analysis device is provided with:

the biopolymer analysis device of claim 1; and

a control unit for controlling the voltages applied to the plurality of 1 st electrodes and the plurality of 2 nd electrodes,

the control unit includes:

an EWOD voltage application circuit that applies the predetermined voltage to the plurality of 1 st electrodes;

a nanopore opening circuit configured to apply a dielectric breakdown voltage of the thin film between the plurality of 1 st electrodes and the plurality of 2 nd electrodes to form a nanopore;

a current measuring circuit for measuring a current flowing between the plurality of 1 st electrodes and the 2 nd electrode; and

and a switch for switching connection between the EWOD voltage application circuit, the nanopore opening circuit, or the current measurement circuit and the plurality of 1 st electrodes.

13. The biopolymer analysis device according to claim 12, wherein,

an insulator is disposed between the EWOD voltage application circuit and the plurality of 1 st electrodes.

14. A biopolymer analysis method comprising:

preparing a biopolymer analysis device, the biopolymer analysis device including: an insulating film containing an inorganic material; a1 st liquid tank and a 2 nd liquid tank partitioned by the film; a plurality of 1 st electrodes disposed in the 1 st liquid bath; and a 2 nd electrode disposed in the 2 nd liquid tank, the 1 st electrodes being configured to be capable of transferring the droplets introduced into the 1 st liquid tank by electrowetting on a dielectric by applying a predetermined voltage;

introducing a hydrophobic liquid into the first liquid tank 1;

introducing the plurality of droplets in the 1 st liquid bath;

delivering the plurality of droplets to a site in contact with the plurality of 1 st electrodes by applying the given voltage to the plurality of 1 st electrodes, the plurality of droplets being insulated from each other by the hydrophobic liquid; and

and introducing an electrolyte solution into the 2 nd liquid tank.

15. The biopolymer analysis method according to claim 14, wherein,

the 1 st liquid tank is also provided with a plurality of 3 rd electrodes,

the plurality of droplets are respectively delivered to the parts contacting with the plurality of No. 1 electrodes and the plurality of No. 3 electrodes,

the biopolymer analysis method further comprises: measuring a current flowing between each of the plurality of No. 3 electrodes and the No. 2 electrode.

16. The biopolymer analysis method according to claim 15, wherein,

the biopolymer analysis method further comprises: applying an insulation breakdown voltage of the thin film between each of the plurality of No. 3 electrodes and the No. 2 electrode to form a nanopore in the thin film.

17. The biopolymer analysis method according to claim 14, wherein,

the plurality of 1 st electrodes and the plurality of 2 nd electrodes are connected to a control unit that controls a voltage applied thereto,

the control unit includes:

an EWOD voltage application circuit that applies the predetermined voltage to the plurality of 1 st electrodes;

a nanopore opening circuit configured to apply a dielectric breakdown voltage of the thin film between the plurality of 1 st electrodes and the plurality of 2 nd electrodes to form a nanopore;

a current measuring circuit for measuring a current flowing through the thin film between the plurality of 1 st electrodes and the plurality of 2 nd electrodes; and

and a switch for switching connection between the EWOD voltage application circuit, the nanopore opening circuit, or the current measurement circuit and the plurality of 1 st electrodes.

18. The biopolymer analysis method according to claim 14, wherein,

the plurality of droplets are droplets comprising a biopolymer,

the biopolymer analysis method further comprises:

forming a nanopore by applying an insulation breakdown voltage of the thin film between the plurality of 1 st electrodes and the 2 nd electrode;

applying a voltage capable of electrophoretically moving the biopolymer between the plurality of 1 st electrodes and the plurality of 2 nd electrodes; and

performing an analysis of the biopolymer based on values of currents flowing between the plurality of 1 st electrodes and the 2 nd electrodes when the biopolymer passes through the nanopore.

19. The biopolymer analysis method according to claim 14, wherein,

the biopolymer analysis method further comprises: determining whether the plurality of droplets are delivered to a desired location.

Technical Field

The present disclosure relates to a biopolymer analysis device, a biopolymer analysis apparatus, and a biopolymer analysis method.

Background

The nanopore (nanopore) device is in a thickness ofIs set to a diameter ofThe electrolyte solution can be passed through the nanopore by bringing the electrolyte solution into contact with both sides of the thin film to generate a potential difference between both ends of the thin film. In this case, when the object to be measured in the electrolytic solution passes through the nanopore, the electrical characteristic, particularly, the resistance value at the periphery of the nanopore changes, and thus the object to be measured can be detected by detecting the change in the electrical characteristic. When the measurement object is a biopolymer, the electrical characteristics of the nanopore peripheral portion change in a pattern according to a monomer sequence pattern (monomer sequence pattern) of the biopolymer. In recent years, methods for analyzing the monomer sequence of a biopolymer using the same have been actively studied.

In particular, it is considered promising to analyze a monomer sequence based on the principle that the amount of change in ion current observed when a biopolymer passes through a nanopore varies depending on the type of monomer. Since the accuracy of analysis of the monomer sequence is determined by the amount of change in ion current, it is preferable that the difference in ion current between the monomers is larger. Unlike the conventional analysis method, this analysis method can directly read the biopolymer without chemical operation involving fragmentation of the biopolymer. The nanopore device is used as a DNA base sequence analysis system (DNA sequencer (sequencer)) when the biopolymer is DNA, and as an amino acid sequence analysis system (amino acid sequencer) when the biopolymer is protein, and is expected to be a system capable of reading a sequence length much longer than that of the conventional system.

In particular, research and development have been actively conducted to put a nanopore into practical use as a DNA sequencer using a current blocking method. The blocking current is a decrease in ion current caused by a decrease in effective cross-sectional area through which an ion can pass, when the biopolymer passes through the nanopore, the biopolymer blocks the nanopore.

As nanopore devices, there are two types, a biological nanopore using a protein having a small pore in the center embedded in a lipid bilayer membrane, and a solid nanopore in which a small pore is processed in an insulating film formed by a semiconductor processing process. In the biological nanopore, a small pore (1.2 nm in diameter and 0.6nm in thickness) of a modified protein (e.g., Mycobacterium smegmatis porin A (MspA)) embedded in a lipid bilayer membrane is used as a biopolymer detection unit to measure the amount of change in ionic current.

On the other hand, among solid nanopores, a structure in which nanopores are formed in a thin film of silicon nitride (SiN) which is a semiconductor material, or a thin film including a monolayer such as graphene or molybdenum disulfide is used as a nanopore device. In a biopolymer analysis method using a solid nanopore, reports have been made so far for measuring the blocking current amount of adenine bases, cytosine bases, thymine bases, and guanine bases of a homopolymer (non-patent documents 1 and 2).

The measurement using the nanopore device has the following three problems. The first problem is that, in order to realize an integrated nanopore device having arrayed parallel channels, it is necessary to insulate the individual channels from each other without leakage of current. If insulation is not performed, the individual channels interfere with each other, and accurate measurement cannot be performed, and independent measurement of each channel becomes difficult.

As a second problem, when the throughput measured by the depletion of the sample during the measurement is decreased, or when a measurement of another sample is desired after a certain sample is sufficiently measured, it is required to extend the effective continuous measurement time by performing smooth sample supply or sample replacement.

As a third problem, in the measurement of biomolecules such as DNA, since a sample collected from a living body is valuable and it is desired to collect only a small amount, it is necessary to be able to measure even a small amount of solution (a small input amount of DNA).

In patent document 1, the following method is attempted in order to realize an integrated nanopore device using a lipid bilayer membrane and a biological nanopore. In a resin flow cell (flow cell) requiring a plurality of parallel wells (wells), a hydrophobic liquid (oil) and an aqueous solution having a material constituting a lipid bilayer membrane are alternately flowed in, so that they spontaneously form individual droplet portions at the bottom of each parallel well and spontaneously form a common solution portion at the top of the well. The integration is achieved by spontaneously forming a lipid bilayer membrane at the interface between each individual droplet portion and the common solution portion, and electrically embedding biological nanopores in the membrane.

In the solid-state nanopore device, unlike a lipid bilayer membrane using self-organization of biological nanopores, a solid-state inorganic thin film formed in advance from an inorganic material is used, and therefore the method as in patent document 1 cannot be applied, and another method (aproach) is required for integration. In non-patent document 3, a method of forming five parallel channels by dividing one inorganic thin film into different segments using a microchannel is attempted.

Further, in non-patent document 4, for an apparatus having 16 independent films, a method of realizing parallel connection by combining an O-ring of an insulating rubber and a resin flow cell is attempted.

In realizing a parallel solid-state nanopore device with high integration, in patent document 2, a method of using a hydrophobic liquid (oil) as an insulator between individual channels is attempted. Such a hydrophobic liquid is realized by a liquid feeding mechanism using a flow path. Patent document 3 describes a method in which an insulating film such as a photosensitive resin is provided as an insulating partition wall between individual channels. Such an insulating film is realized by a liquid feeding mechanism using a pressure bonding method.

As described above, it is common in the integrated nanopore device to provide a common solution reservoir on one side of the membrane and a plurality of independent individual solution reservoirs on the other side. Such a structure is a basic construction in an integrated nanopore device.

Prior art documents

Patent document

Patent document 1: international publication No. 2014/064443

Patent document 2: japanese patent No. 6062569

Patent document 3: japanese laid-open patent publication No. 2018-96688

Non-patent document

Non-patent document 1: feng J., et al, Identification of single nucleotides in MoS2 nanopores, Nat. nanotechnol.10, 1070-1076 (2015).

Non-patent document 2: goto Y, et al, Identification of four single-stranded DNA macromolecules with a solid-state nanopore in alkaline CsCl solution Nanoscale 10, 20844-.

Non-patent document 3: tahvildari R, et al, Integrating nanopores sensors with microfluidic channel arrays using controlled breakdown, Lab on a Chip 15, 1407-.

Non-patent document 4: yanagi I., et al, Multichannel detection of ionic currents through integrated Si3N4membranes Lab on a Chip 16, 3340-.

Disclosure of Invention

Problems to be solved by the invention

However, in the conventional integrated solid-state nanopore system, it is difficult to achieve both the collective injection of a solution into a plurality of independent individual solution cells and the replacement of a solution (sample) in an individual solution cell while maintaining the insulation between the channels. Although the solution exchange is easy by using a flow path such as a flow cell, a special liquid feeding device such as a jig or a pump is required to collectively inject the solution into the individual solution tanks, and the apparatus becomes complicated. This problem becomes remarkable when the integration degree becomes large and the flow path becomes minute.

Since the separate solution tank formed by the pressure welding method as in patent document 3 is a closed space, it is difficult to replace the original solution.

Further, in the conventional method, since a volume amount of the solution larger than a volume of the solution in the individual solution tank is required to dispose the solution in the individual solution tank, there is also a problem that it is difficult to measure a sample with a small volume amount of the solution.

Accordingly, the present disclosure provides a technique for achieving both automatic batch injection of a solution into a plurality of individual solution tanks and automatic replacement of a solution in an individual solution tank while maintaining insulation between parallel channels.

Means for solving the problems

In order to solve the above problems, a biopolymer analysis device of the present disclosure includes: an insulating film containing an inorganic material; a1 st liquid tank and a 2 nd liquid tank partitioned by the film; a plurality of 1 st electrodes disposed in the 1 st liquid bath; and a 2 nd electrode disposed in the 2 nd liquid tank, a hydrophobic liquid and a plurality of droplets are introduced into the 1 st liquid tank, the plurality of 1 st electrodes are configured to be capable of transferring the plurality of droplets introduced into the 1 st liquid tank by electrowetting on a dielectric by applying a predetermined voltage, the plurality of droplets are transferred to a portion in contact with the plurality of 1 st electrodes, and are insulated from each other by the hydrophobic liquid.

Further features relevant to the present disclosure will be apparent from the description of the present specification and the accompanying drawings. The embodiments of the present disclosure are achieved and realized by the elements and combinations of various elements, the following detailed description, and the appended claims.

The description of the present specification is merely exemplary and does not limit the claims or application examples of the present disclosure in any way.

Effects of the invention

According to the present disclosure, it is possible to achieve both automatic batch injection of a solution into a plurality of individual solution tanks and automatic replacement of a solution in an individual solution tank while maintaining insulation between parallel channels.

Problems, structures, and effects other than those described above will be apparent from the following description of the embodiments.

Drawings

Fig. 1 is a schematic diagram showing a biopolymer analysis device of a single channel according to a reference example.

Fig. 2 is a schematic diagram showing a biopolymer analysis device with parallel channels according to a reference example.

Fig. 3A is a schematic diagram showing a biopolymer analysis device according to embodiment 1.

Fig. 3B is a schematic diagram showing the biopolymer analysis device after nanopore aperturing.

Fig. 4 is a schematic view showing another biopolymer analysis device according to embodiment 1.

Fig. 5 is a schematic view showing another biopolymer analysis device according to embodiment 1.

Fig. 6 is a flowchart illustrating the biopolymer analysis method according to embodiment 1.

Fig. 7 is a schematic diagram showing a biopolymer analysis device according to embodiment 2.

FIG. 8A is a plan view of the first liquid tank 1 of the biopolymer analysis device according to embodiment 2.

Fig. 8B is a plan view showing a state where a droplet is transported.

Fig. 8C is a plan view showing a state where all the droplets are arranged at desired positions.

Fig. 9 is a schematic diagram showing a biopolymer analysis device according to embodiment 3.

Fig. 10A is a schematic view showing a state where a hydrophobic liquid remains on the surface of the thin film.

Fig. 10B is a schematic diagram showing the configuration of the sacrificial layer of embodiment 4.

Fig. 10C is a schematic view showing another biopolymer analysis device according to embodiment 4.

Fig. 11 is a schematic view showing a biopolymer analysis device according to embodiment 5.

Fig. 12 is a schematic view showing another biopolymer analysis device according to embodiment 5.

Fig. 13 is a schematic diagram showing a biopolymer analysis device according to embodiment 6.

Fig. 14A is a schematic diagram showing a biopolymer analysis device according to embodiment 7.

Fig. 14B is a schematic diagram showing a biopolymer analysis device according to embodiment 7.

Fig. 15 is a schematic view showing a biopolymer analysis device according to embodiment 8.

Detailed Description

Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. In addition, the drawings illustrate specific embodiments following the principles of the present disclosure, but they are for understanding the present disclosure and are in no way to be construed as limiting the disclosure.

The structure of the apparatus for analyzing a biopolymer differs depending on the method of introducing the biopolymer into the nanopore, but in the present specification, a description is given of a method of introducing the biopolymer into the nanopore by electrophoresis as an example. The term "biopolymer" as used herein means DNA or RNA containing nucleic acids as monomers, or protein or polypeptide containing amino acids as monomers.

[ reference example ]

Fig. 1 is a schematic diagram showing a biopolymer analysis device 100 having a single nanopore tunnel according to a reference example. As shown in fig. 1, a biopolymer analysis device 100 includes a thin film 102 having a nanopore 101, a1 st liquid tank 104A and a 2 nd liquid tank 104B that contain an electrolyte solution 103, and electrodes 105A and 105B.

The electrodes 105A and 105B are connected to an ammeter 106 and a power source 107, and a voltage is applied to the electrodes 105A and 105B by the power source 107. The voltage application based on the power supply 107 is controlled by the computer 108.

The ammeter 106 measures an ion current (blocking current) flowing between the electrode 105A and the electrode 105B. Although not shown, the ammeter 106 includes an amplifier and an analog/digital converter that amplify the current flowing between the electrodes 105A and 105B. The ammeter 106 is connected to the computer 108, and the analog/digital converter outputs the detected value of the ion current to the computer 108 as a digital signal.

The computer 108 acquires monomer sequence information of the biopolymer 1 based on the value of the ion current (blocking current).

Fig. 2 is a schematic diagram showing a biopolymer analysis device 200 as an array device having nanopore channels connected in parallel according to a reference example. The array device is a device provided with a plurality of individual solution tanks partitioned by partition walls. As shown in fig. 2, the biopolymer analysis device 200 is different from the biopolymer analysis device 100 of fig. 1 in that it has a plurality of 2 nd liquid cells 104B electrically insulated by tapered layers 102B as partitions, and one electrode 105B is provided in each of the plurality of 2 nd liquid cells 104B.

Thus, the 1 st liquid tank 104A serves as a common solution tank, and the 2 nd liquid tank 104B serves as a plurality of individual solution tanks, and a plurality of independent channels are formed. The electrode 105A is a common electrode, and the electrode 105B is an individual electrode.

[ embodiment 1]

< example of configuration of biopolymer analysis device >

Fig. 3A is a schematic diagram showing a biopolymer analysis device 300 according to embodiment 1. As shown 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, a1 st liquid cell 104A, a 2 nd liquid cell 104B, a common electrode 105 (a 2 nd electrode), and a substrate 113 having a plurality of individual electrodes 112 (a plurality of 1 st 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) and silicon oxide (SiO)₂) Silicon oxynitride (SiON), hafnium oxide (HfO)₂) Molybdenum disulfide (MoS)₂) Graphene, and the like. The thickness of the film 102 can be set, for exampleAccording to circumstances, can be set asOrAs an example can be set to about 5 nm.

Although not shown, the common electrode 105 can be connected to the ammeter 106, the power supply 107, and the computer 108 (control unit) shown in fig. 1 and 2 via wires, and the plurality of individual electrodes 112 can be connected to the ammeter via wires inside the substrate 113.

As will be described later, the computer 108 controls the application of voltages 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 individual electrodes 112 or between the individual electrodes 112 and the common electrode 105, and determines the position of the droplet 110, whether or not a leak is generated between the droplets 110, and whether or not a nanopore is formed in the thin film 102, based on the electrical characteristics such as the measured current value. The computer 108 includes a storage unit (not shown) and stores the measured current value and the result of the determination in the storage unit.

The individual electrodes 112 are embedded in a substrate 113, and the substrate 113 constitutes a part of the 1 st liquid tank 104A. The substrate 113 may be made of any material as long as it can be mounted with circuit wiring, and for example, a PWB substrate such as glass epoxy resin, a PCB substrate, or the like is used. Alternatively, the substrate 113 may be a transparent substrate such as a glass substrate.

In the 1 st liquid tank 104A, a plurality of droplets 110 and a hydrophobic liquid 111 are introduced. Each droplet 110 is electrically insulated from adjacent droplets 110 by a hydrophobic liquid 111, independent of each other. Further, the plurality of droplets 110 are in contact with the individual electrodes 112, respectively, and thus electrical operations such as application of a voltage can be performed on each droplet 110.

The individual electrodes 112 transport the droplets 110 to a desired position by electrowetting on Dielectric (EWOD: Electro Wetting 0n direct) by applying a given voltage between adjacent individual electrodes 112. In fig. 3A, a state where the droplets 110 are respectively transported to positions in contact with the individual electrodes 112 is shown, and the droplets 110 are separated from each other by the hydrophobic liquid 111, being insulated from each other. Thereby, a plurality of individual solution tanks (a plurality of channels) are formed.

The application of the voltage for EWOD feeding (predetermined voltage) for operating the individual electrode 112 as an EWOD electrode is controlled by the computer 108. The voltage for EWOD transport can be set, for example, to 0 to 100V, typically, within a range of 10 to 50V. The voltage value is changed every time according to the diameter and viscosity of the droplet 110, the contact angle formed by the droplet 110, the hydrophobic liquid 111, and the individual electrode 112, the electrode size, and the like, and is adjusted as appropriate.

The individual electrode 112 is also used to open the nanopore 101 or measure an 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 2 nd liquid tank 104B, and a common electrode 105 is disposed so as to be in contact with the electrolyte solution 103. Here, the plurality of droplets 110 and the electrolyte solution 103 are aqueous solutions containing electrolytes, and may contain a biopolymer to be analyzed.

The capacity of the electrolyte solution 103 can be set to micro-upgrade or milliupgrade. The volume of the droplet 110 can be nano-scaled or micro-scaled.

The 1 st liquid tank 104A and the 2 nd liquid tank 104B that store the measurement solution that is in contact with the thin film 102 can be appropriately provided with materials, shapes, and sizes that do not affect the measurement of the ion current.

The individual electrodes 112 and the common electrode 105 can be made of a material capable of undergoing an electron transfer reaction (faraday reaction) with the electrolyte in the liquid droplets 110 and the electrolyte solution 103, and examples thereof include silver halide and alkali silver halide. From the viewpoint of potential stability and reliability, silver or silver/silver chloride can be used in particular.

The material of the individual electrodes 112 and the common electrode 105 may be a material that becomes a polarizing electrode, and for example, gold, platinum, or the like may be used. In this case, in order to ensure a stable ion current, for example, a substance capable of assisting an electron transfer reaction, such as potassium ferricyanide or potassium ferrocyanide, may be added to the measurement solution. Alternatively, for example, a substance capable of undergoing an electron transfer reaction such as ferrocene can be immobilized on the surface of the polarizing electrode.

The individual electrodes 112 and the common electrode 105 may be formed of the above-described material, or the surface of a base material (copper, aluminum, or the like) may be covered with the above-described material. The shapes of the individual electrodes 112 and the common electrode 105 are not particularly limited, and may be a shape having a large surface area in contact with the measurement solution. The individual electrodes 112 and the common electrode 105 are connected to a wire, and transmit an electric signal to a measurement circuit.

The hydrophobic liquid 111 is an insulating liquid separated from the aqueous phase, and a liquid having a high affinity with the biopolymer can be used in some cases. Examples of the hydrophobic liquid 111 include silicone oil, fluorine-based oil, and mineral oil. Such liquids are also frequently used in techniques such as PCR, digital PCR, and the like. Further, since the hydrophobic liquid 111 is used for the transportation of the EWOD-based droplets 110, a liquid having low viscosity and high fluidity can be used as the hydrophobic liquid 111.

Although not shown, the 1 st liquid tank 104A and the 2 nd liquid tank 104B each have an injection port for injecting a liquid into the tank and a discharge port for discharging the liquid from the tank.

< method for Forming Nano pores >

Fig. 3B is a schematic diagram showing the biopolymer analysis device 300 in a state where the nanopore 101 is formed in the thin film 102. In the state of the structure of fig. 3A, since the nanopore 101 is not provided, the biopolymer cannot be analyzed. Therefore, the nanopore 101 can be formed by applying a voltage value equal to or higher than the insulation breakdown voltage of the thin film 102 between the plurality of individual electrodes 112 and the common electrode 105.

The method for forming the nanopore 101 in 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. The method of forming the nanopore 101 can be, for example, the method described in "Itaru Yanagi et al, sci. rep.4, 5000 (2014)".

The film 102 is made of Si₃N₄The formation of the nanopore 101 by voltage application in the case of the configuration can be performed by, for example, the following steps. Firstly, the methodBy Ar/O₂Plasma (plasma) (manufactured by sameck corporation, Samco Inc.) was applied to Si at 10WW, 20sccm, 20Pa, and 45sec₃N₄The film 102 thus constituted is hydrophilized. Next, a biopolymer analysis device 300 provided with a thin film 102 is installed in the flow cell. Then, the individual electrode 112 and the common electrode 105 were introduced into the 1 st liquid tank 104A and the 2 nd liquid tank 104B, respectively, and 1M CaCl was contained₂And 1mM Tris-10mM EDTA in pH7.5 electrolyte solution, i.e., droplet 110, to the 1 st liquid tank 104A, and the 2 nd liquid tank 104B is filled with the electrolyte solution.

The voltage is applied not only when the nanopore 101 is formed, but also when the ion current flowing through the nanopore 101 after the nanopore 101 is formed is measured. Here, the 1 st liquid tank 104A located on the GND electrode side is referred to as a cis tank, and the 2 nd liquid tank 104B located on the variable voltage side is referred to as a trans tank. Voltage V to be applied to the cis cell side electrode_cisSet to 0V, and apply a voltage V to the electrodes on the trans tank side_trans. Voltage V_transFor example, by a Pulse Generator (41501B SMUAND Pulse Generator Expander, manufactured by Agilent Technologies).

The current value after the pulse application can be read by an ammeter 106(4156B PRECISION SEMICONDUCTOR ANALYZER inductor, manufactured by Agilent Technologies). The process of applying a voltage to form the nanopore 101 and the process of reading an ion current value are controlled by, for example, a homemade program (Excel VBA, Visual Basic for Applications) stored in a memory unit of the computer 108. The current value condition (threshold current) is selected according to the diameter of the nanopore 101 formed before the pulse voltage is applied, the diameter of the nanopore 101 is sequentially increased, and the diameter as a target is obtained.

The diameter of the nanopore 101 can be estimated from the ion current value. The criterion for selecting the condition is, for example, that the material of the thin film 102 is Si₃N₄And the thickness of the thin film 102 was 5nm, as shown in Table 1. Here, the nth pulse voltage application time t_n(wherein n > 2 is an integer.) is defined by the following formula.

[ numerical formula 1]

t_n＝10^{-3+(1/6)(n-1)}-10^{-3+(1/6)(n-2)}For n＞2

[ Table 1]

TABLE 1 Voltage application conditions

Formation of the nanopore 101 can be performed by electron beam irradiation by TEM in addition to a method of applying a pulse voltage (a.j.storm et a1., nat.mat.2 (2003)).

The size of the nanopore 101 can be selected according to the type of biopolymer to be analyzed, and can be set to, for example, 0.9nm to 100nm, and in some cases, 0.9nm to 50 nm. Specifically, the size of the nanopore 101 is 0.9nm or more and 10nm or less. For example, the nanopore 101 used for analyzing a single-stranded DNA having a diameter of about 1.4nm can have a diameter of, for example, 0.8nm to 10nm or 0.8nm to 1.6 nm. For example, the nanopore 101 used for analyzing a double-stranded DNA having a diameter of about 2.6nm can have a diameter of 3nm to 10nm or 3nm to 5 nm.

The 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, and in some cases, three times or more, or five times or more, the monomer unit constituting the biopolymer. For example, when the biopolymer is a nucleic acid, the depth of the nanopore 101 is set to a size of three or more bases, for example, about 1nm or more. This makes it possible to control the shape and the moving speed of the biopolymer and to cause the biopolymer to enter the nanopore 101, thereby enabling highly sensitive and highly accurate analysis. The shape of the nanopore 101 is substantially circular, but may be elliptical or polygonal.

Immediately before the user analyzes the biopolymer using the biopolymer analysis device 300, as shown in fig. 3B, the liquid droplets 110 are transported to a position in contact with the individual electrodes 112, and are insulated from each other by the hydrophobic liquid 111, and the nanopores 101 are provided in the thin film 102 by electrical operation, whereby the nanopores 101 having good quality can be provided at all times.

Further, the biopolymer analysis device 300 may be provided to the user in a state where the droplet 110 and the hydrophobic liquid 111 are transported to the position shown in fig. 3A, or may be provided to the user in a state where only the hydrophobic liquid 111 is introduced into the 1 st liquid tank 104A, and the droplet 110 may be transported to the position shown in fig. 3A by applying an EWOD transport voltage to the individual electrode 112 by the user's operation. Further, the biopolymer analysis device 300 may be provided to the user in a state where both the 1 st liquid tank 104A and the 2 nd liquid tank 104B are empty. In this case, after the hydrophobic liquid 111 is filled in the 1 st liquid tank 104A by the user's operation, the droplet 110 is transferred by applying the voltage for EWOD transfer to the individual electrode 112, and the electrolyte solution 103 is introduced into the 2 nd liquid tank 104B, thereby bringing the state shown in fig. 3A.

< example of another configuration of biopolymer analysis device >

Fig. 4 is a schematic diagram showing another biopolymer analysis device 400 according to embodiment 1. The biopolymer analysis device 400 has a structure in which the structure of the present embodiment (fig. 3) is adopted for a typical solid-state nanopore device used for analyzing a biopolymer in a current blocking mode. As shown in fig. 4, biopolymer analysis device 400 has thin film 102A made of an inorganic material, tapered layer 102B disposed on one side of thin film 102A, and sacrificial layer 102C disposed on the other side of thin film 102A. In addition, the thin film 102A, the tapered layer 102B, and the sacrificial layer 102C may be collectively referred to as a "thin film".

As the material of the taper layer 102B and the sacrifice layer 102C, silicon (Si) is generally used in consideration of mass productivity. The tapered layer 102B is formed by anisotropic etching of a silicon wafer, for example. The sacrificial layer 102C has a plurality of (three in fig. 4) etching holes (projections) formed by etching, for example, a silicon wafer, at positions facing the individual electrodes 112, and thereby the thin films 102A are exposed at a plurality of portions and arrayed. Further, the sacrificial layer 102C supports the film 102A by stress. The structure of such a solid-state nanopore device is described, for example, in U.S. patent No. 5795782, "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 ", etc.

The size of the thin film 102A exposed in the droplet 110 needs to be an area where it is difficult to form two or more nanopores 101 when forming the nanopores 101 by applying a voltage, and an area that is allowable in terms of strength. The area is, for example, about 100 to 500nm, and the thickness of the nano-pores 101, which can form an effective film thickness corresponding to a single base, is preferably about 3 to 7nm in order to achieve a single base resolution of DNA.

As shown in fig. 4, in the case of a structure in which a plurality of individual solution reservoirs are arrayed, exposed portions of the thin film 102 in which the nanopores 101 are formed can be regularly arranged. The intervals between the exposed portions of the thin film 102A can be set to, for example, 0.1mm to 10mm or 0.5mm to 4mm, depending on the electrodes used and the capability of the electrical measurement system.

Fig. 5 is a schematic diagram showing another biopolymer analysis device 500 according to embodiment 1. As shown in fig. 5, the biopolymer analysis device 500 is different from the biopolymer analysis device 400 shown in fig. 4 in that a plurality of tapered layers 102B are provided. Such structures are described, for example, in "Yanagi, et al, Lab on a Chip, 16, 3340-.

< method for analyzing biopolymer >

Hereinafter, a method for continuously performing nanopore formation and biopolymer analysis using a biopolymer analysis device before nanopore formation will be described. In the biopolymer analysis method according to the present embodiment, any one of the biopolymer analysis devices 300 to 500 shown in fig. 3A, 4, and 5 may be used, and the common electrode 105 and the plurality of individual electrodes 112 may be connected to the ammeter 106, the power supply 107, and the computer 108 shown in fig. 1 and 2. Furthermore, a biopolymer analysis device in which the 1 st liquid tank 104A and the 2 nd liquid tank 104B are empty is used.

Fig. 6 is a flowchart showing a biopolymer analysis method using a biopolymer analysis device according to the present embodiment. First, in step S1, the user introduces the hydrophobic liquid 111 from an inlet (not shown) of the 1 st liquid tank 104A (on the side of the individual electrode 112), and fills the 1 st liquid tank 104A with the hydrophobic liquid 111.

In step S2, the user inputs an instruction to start the operation to the computer 108, and sequentially injects the plurality of droplets 110 into an injection port (not shown) of the 1 st liquid tank 104A. Here, each of the plurality of droplets 110 is an electrolyte solution for forming a nanopore.

Upon receiving the operation start instruction, the computer 108 applies an EWOD transport voltage to the individual electrodes 112 via the power supply 107, and transports a plurality of droplets 110 such that each droplet 110 is disposed at a position in contact with one individual electrode 112. At this time, the hydrophobic liquid 111 prevents the droplets 110 from contacting each other, and electrically insulates the droplets 110 from each other. Thereby, a plurality of independent individual solution reservoirs (a plurality of channels) each having one individual electrode 112 and one droplet 110 are formed.

In step S3, the computer 108 detects the position at which the plurality of droplets 110 are conveyed. Next, in step S4, the computer 108 determines whether the droplet 110 has moved to a desired position. The method of determining the position of the droplet 110 will be described later. If the droplet 110 does not reach the desired position ("no"), the process returns to step S2, and the computer 108 repeats the transport of the droplet 110 until the desired position is reached.

After the droplet 110 reaches the desired position (yes in step S4), in step S5, the computer 108 applies a voltage for reading the leak current between the individual electrodes 112 of the adjacent channels, and measures the leak current value.

In step S6, the computer 108 determines whether or not the measured leakage current value is smaller than a predetermined threshold value.

If the leakage current value is equal to or greater than the threshold value (no in step S6), the channel does not maintain electrical independence, and therefore, the process returns to step S2, and the computer 108 attempts measurement of the leakage current from the transport of the droplet 110 again until the leakage current value is less than the threshold value. Alternatively, instead of returning to step S2, 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 unit.

If the leakage current value is smaller than the threshold value (yes in step S6), it can be determined that the channel is a good channel, and the process proceeds to step S7.

After the droplets 110 move to all the channels and the electrical independence is confirmed, the user introduces the electrolyte solution 103 into the 2 nd liquid tank 104B in step S7.

In step S8, the computer 108 applies a voltage equal to or higher than the dielectric breakdown voltage of the film 102 between each individual electrode 112 and the common electrode 105 to electrically open the nanopore 101. The computer 108 directly applies a voltage for nanopore characteristic determination between each individual electrode 112 and the common electrode 105, and measures the current-voltage characteristic of the nanopore 101. Here, when the measured current value falls within a range of a desired current value, that is, within a range of a desired nanopore diameter, it is determined that a good nanopore 101 is 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 the positional information of the discarded channel in the storage section so that the droplet containing the sample does not move to the discarded channel. This can prevent the loss of the sample.

The droplet 110 transferred to the individual electrode side by the above operation is an electrolyte solution for opening a nanopore, and therefore needs to be replaced with a solution for measuring a sample. In step S9, the computer 108 applies an EWOD transport voltage to the individual electrode 112, and transports the droplet 110 as each nanopore-perforated solution to the discharge port of the 1 st liquid tank 104A, and moves the droplet to a waste liquid tank (not shown) connected to the discharge port.

Then, the user injects a droplet (sample solution) for measuring a sample containing a biopolymer from the injection port of the first liquid tank 104A, and the computer 108 applies an EWOD transfer voltage to the individual electrode 112 to move the sample solution to a site where a good nanopore 101 is formed.

After the entire sample solution is transferred, in step S10, the computer 108 applies a voltage for sample measurement between each individual electrode 112 and the common electrode 105 to measure the sample.

Further, when the sample is replaced, the same operation as step S9 is performed. Specifically, the computer 108 applies an EWOD transport voltage to the individual electrode 112, transports the measured sample solution to the discharge port of the 1 st liquid tank 104A, and moves the sample solution to a waste liquid tank connected to the discharge port. Then, the user introduces a new sample solution from the inlet of the 1 st liquid tank 104A, and the computer 108 applies an EWOD transfer voltage to the individual electrode 112 to transfer the new sample solution. Thus, the solution in each individual solution tank can be smoothly replaced by EWOD.

< method for determining position of droplet >

Next, a method of detecting the position of the droplet 110 in steps S3 and S4 will be described. Whether the droplet 110 reaches a desired position can be detected by various methods. For example, the inside of the 1 st liquid tank 104A can be optically observed as an image by using a transparent substrate and a transparent electrode as the individual electrode 112 and the substrate 113, and providing an observation device (a mechanism for determining whether or not the plurality of droplets are transported to a desired position) such as a microscope above the individual electrode 112 and the substrate 113. The observation device is configured to be able to transmit image data of an observed portion captured to the computer 108, and the computer 108 is able to determine the position of the droplet 110 based on the image data.

On the other hand, when the individual electrodes 112 and the substrate 113 are made of an opaque material, the droplet 110 cannot be observed as an image. In this case, the position of the droplet 110 can be determined by using an electrical method without using the optical method as described above. The droplets 110 transported by the biopolymer analysis device of the present embodiment contain an electrolyte, and therefore are electrically conductive. Therefore, by applying an electrical operation between the individual electrodes 112 or between the individual electrodes 112 and the common electrode 105 and investigating the presence or absence of a change in the electrical reaction, it can be determined whether the droplet 110 is in contact with the individual electrodes 112 (at the position of the individual electrode 112).

Further, the impedance characteristics at the time of alternating current application differ depending on whether the hydrophobic liquid 111 containing an electrolyte or the electrolyte solution is in contact with the individual electrode 112, for example. Therefore, by applying an alternating current to the individual electrode 112 and measuring the impedance, it is possible to know whether or not the liquid droplet 110 is in contact with the individual electrode 112.

Alternatively, the position of the droplet 110 can be determined by measuring the current value between the individual electrode 112 and the common electrode 105 to examine the resistance characteristics. For example, in a state where the hydrophobic liquid 111 is in contact with the individual electrodes 112 and the thin film 102, the individual electrodes 112 and the common electrode 105 are completely insulated from each other by the high insulation property of the hydrophobic liquid 111, and thus the observed current value is 10^-13～10^-14A is below. On the other hand, in a state where the electrolyte solution such as the droplet 110 is in contact with the individual electrode 112 and the thin film 102, since the electrolyte solution is a low resistance body, 10 can be observed between the individual electrode 112 and the common electrode 105 even before the nanopore 101 is opened^-11～10^-12The current value of A. Such a current value is observed, for example, in "Scientific Reports, 5, 14656, 2015, Yanagi, et al. In this way, by detecting the difference in current value, it is possible to know whether or not the droplet 110 is in contact with the individual electrode 112 and the thin film 102, and thus the position of the droplet 110 can be determined.

< technical effects >

As described above, in embodiment 1, by applying an EWOD feeding voltage to the individual electrodes 112 and automatically moving the plurality of droplets 110 to desired positions, it is possible to collectively inject the solution 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 hydrophobic liquid 111, and thus are electrically independent. In addition, when the solution is replaced, the new droplet 110 may be transported to a desired position by simply transporting the droplet 110 by the EWOD and discarding the droplet, and thus the solution can be replaced smoothly. Therefore, it is possible to achieve both the collective injection of the solution into the plurality of independent individual solution tanks and the replacement of the solution in the individual solution tank while maintaining the insulation between the parallel channels. Further, since a liquid feeding device for feeding and replacing the solution is not required, it is possible to avoid an increase in size of the device and an increase in installation cost.

EWOD is effective even when the degree of integration is high, that is, when the size of the component is small. In particular, EWOD can deliver even a small droplet of several μ L to several nL, and thus can measure a sample with a small droplet amount.

Further, the biopolymer analysis device of the present embodiment can integrate independent individual solution tanks. Therefore, the measurement can be performed simultaneously for different types of samples. For example, samples of different types can be measured simultaneously by preparing a certain droplet as a solution of sample a and another droplet as a solution of sample B, and transporting the prepared droplets to appropriate positions. In addition, when the biopolymer analysis device of the present embodiment is used as a DNA sequencer, for example, a sample a having a genetic variation a and a sample B having a genetic variation B can be simultaneously measured in one device. The same applies to a gene detection method based on hybridization (hybridization) in which a probe is immobilized. Alternatively, DNA sequencing, the hybridization detection method, and the like may be performed simultaneously. By integrating the individual solution tanks in this manner, the throughput of measurement can be improved.

[ 2 nd embodiment ]

In general, when a droplet is transported by EWOD, an insulator (dielectric) may be provided on the surface of an electrode in order to extract charges from the surface of the droplet and polarize the charges to improve wettability to the surface of the electrode. However, when the surface of the individual electrode 112 of embodiment 1 is provided with an insulator, it is difficult to measure the current with a high insulation resistance, and it is impossible to analyze the biopolymer using the individual electrode 112.

In order to solve such a problem, in embodiment 2, as individual electrodes, two or more electrodes for current measurement and an electrode for EWOD are provided for each droplet.

< example of configuration of biopolymer analysis device >

Fig. 7 is a schematic diagram showing a biopolymer analysis device 700 according to embodiment 2. The structure of the substrate 113 of the biopolymer analysis device 700 is different from the biopolymer analysis device 400 shown in fig. 4. Therefore, the structure other than the substrate 113 will not be described.

As shown in fig. 7, a plurality of individual electrodes 112 (a plurality of 3 rd electrodes) for current measurement and a plurality of EWOD electrodes 114 (a plurality of 1 st electrodes) are embedded in a substrate 113. The individual electrodes 112 are disposed at positions facing the exposed portions of the thin film 102A, respectively. An insulator 115 is provided on the inner surface of the EWOD electrode 114. As described later, the plurality of EWOD electrodes 114 are arranged so as to form a passage (lane) for transporting each droplet 110 at a position in contact with each individual electrode 112.

Fig. 7 shows a state where droplets 110 are transported to a desired position, and each droplet 110 is in contact with at least one individual electrode 112 and a plurality of electrodes 114 for EWOD surrounding the individual electrode. In this way, by providing the electrode for current measurement and the electrode for EWOD as different applications, EWOD feeding and current measurement can be performed without any problem.

< method for analyzing biopolymer >

The biopolymer analysis method of the present embodiment is substantially the same as that of embodiment 1 (fig. 6), but differs from embodiment 1 in that, in the droplet transport in steps S2 and S9, an EWOD transport voltage is applied not to individual electrode 112 but to EWOD electrode 114.

Fig. 8A is a top view of biopolymer analysis device 700. As shown in fig. 8A, 16 individual electrodes 112 for current measurement in total of 4 columns × 4 rows are arranged on a substrate 113, and a plurality of EWOD electrodes 114 are arranged around each individual electrode 112. In this way, the plurality of EWOD electrodes 114 form a passage for transporting the droplet 110, and the droplet 110 can be smoothly transported. The individual electrodes 112 are disposed above the exposed portions of the film 102. When each individual electrode 112 is a transparent electrode, the thin film 102 can be viewed from above the individual electrode 112 as shown in fig. 8A. The state shown in fig. 8A is after the hydrophobic liquid 111 is introduced in step S1 (fig. 6) described in embodiment 1.

Fig. 8B and 8C are plan views of biopolymer analysis device 700 showing the state where droplet 110 is transported. As described above, the droplet 110 is transported by applying an EWOD transport voltage to the EWOD electrode 114. As shown in fig. 8B, for example, when the droplets 110 transported through the flow channel of the flow cell are introduced into the 1 st liquid tank 104A and brought into contact with the EWOD electrodes 114 to which the EWOD transport voltage is applied, the droplets 110 can be discretely transported by an amount corresponding to each electrode. Finally, one droplet 110 is arranged between the membrane 102 and the individual electrode 112. By repeating this operation in the same manner, as shown in fig. 8C, the liquid droplets 110 can be arranged between the exposed portions of all the thin films 102 and the individual electrodes 112.

The number and arrangement of the individual electrodes 112 and the EWOD electrodes 114 are not limited to those shown in fig. 8A to 8C, and can be changed as appropriate. For example, when the channels are highly integrated, the individual electrodes 112 may be provided in units of several hundred to several thousand or more.

< technical effects >

As described above, in the present embodiment, the individual electrode 112 for current measurement and the electrode 114 for EWOD are provided in the 1 st liquid tank 104A. Thus, even if the insulator 115 is provided on the surface of the EWOD electrode 114, formation of a nanopore and measurement of current can be performed using the individual electrode 112 without any problem.

[ embodiment 3 ]

As described above, when the surface of the individual electrode 112 of embodiment 1 is provided with an insulator (dielectric), it is difficult to measure the current with a high insulation resistance, and it becomes impossible to analyze the biopolymer using the individual electrode 112.

In order to solve such a problem, in embodiment 3, a circuit for EWOD feeding, a circuit for nanopore drilling, and a circuit for current measurement are connected to each individual electrode 112, and the voltage applied to the individual electrode 112 is controlled by switching these circuits.

< example of configuration of biopolymer analysis device >

Fig. 9 is a schematic diagram showing a biopolymer analysis device 800 according to embodiment 3. The structure of the biopolymer analysis device 800 is substantially the same as the biopolymer analysis device 400 of fig. 4 described in embodiment 1, but a control circuit 121 (control unit) is connected to each individual electrode 112 (a plurality of 1 st electrodes) via a wire. As shown in fig. 9, the control circuit 121 is provided with an EWOD feeding circuit 116, a nanopore drilling circuit 117, a current meter measuring circuit 118, and a plurality of switches 122 for switching these circuits. The control circuit 121 is connected to the computer 108 (control unit), and the computer 108 controls switching of the switch 122 and application of voltage using the circuits 116 to 118.

By providing a circuit having a structure such that charges are appropriately extracted from droplets, for example, a capacitor 123 (insulator) between the EWOD transport circuit 116 and the individual electrode 112, EWOD transport can be appropriately performed without providing an insulator on the surface of the individual electrode 112. In addition, one EWOD transmission circuit 116 may be provided in common to all the individual electrodes 112.

< method for analyzing biopolymer >

The method of analyzing a biopolymer in the present embodiment is substantially the same as that in embodiment 1 (fig. 6), but differs from embodiment 1 in that the computer 108 changes the voltage applied to the individual electrodes 112 by switching the switch 122. Therefore, only the differences from embodiment 1 will be described.

In step S2, the computer 108 switches the switch 122 to connect the EWOD feeding circuit 116 to each individual electrode 112, and applies an EWOD feeding voltage to each individual electrode 112.

In step S5, the computer 108 switches the switch 122, connects the current measuring circuit 118 to each individual electrode 112, and applies a voltage for leak current reading between the individual electrodes 112 of the adjacent channels to measure a leak current value.

In step S8, the computer 108 switches the switch 122, connects the nanopore opening circuit 117 to each individual electrode 112, and applies a voltage equal to or higher than the breakdown voltage of the thin film 102 between each individual electrode 112 and the common electrode 105 to electrically open the nanopore 101.

In step S9, the computer 108 switches the switch 122 to connect the EWOD feeding circuit 116 to each individual electrode 112. Next, an EWOD transport voltage is applied to the individual electrode 112, and the droplets 110 as each nanopore-opened solution are transported to the discharge port of the 1 st liquid tank 104A and moved to a waste liquid tank (not shown) connected to the discharge port.

In step S10, the computer 108 switches the switch 122, connects the current measuring circuit 118 to each individual electrode 112, and applies a sample measuring voltage between each individual electrode 112 and the common electrode 105 to measure the sample.

< technical effects >

As described above, in the present embodiment, the EWOD feeding circuit 116, the nanopore opening circuit 117, and the current measuring circuit 118 are connected to the plurality of individual electrodes 112, and the circuit connected to the individual electrodes 112 is switched by the switch 122. Thus, the droplet 110 can be transported, the nanopore can be formed, and the current value can be measured only by the individual electrode 112 and the common electrode 105 without separately providing an EWOD electrode, and therefore, the number of channels per unit area of the biopolymer analysis device can be increased as compared with embodiment 2.

[ 4 th embodiment ]

As shown in fig. 4 and 5, a solid-state nanopore device has a structure in which a sacrificial layer 102C, which is a flat surface, is provided on one side of a thin film 102A, and a tapered layer 102B, which is a tapered surface, is provided on the other side. However, the sacrificial layer 102C has a structure (etching hole) in which only a specific region is cut by chemical etching or dry etching in order to expose the thin film 102A.

According to the structure of the biopolymer analysis device, the hydrophobic liquid 111 remains in the etching hole, and the droplet 110 cannot enter the etching hole, resulting in a problem of a defective channel.

Fig. 10A is a schematic view showing a state where the hydrophobic liquid 111 remains in the etching holes 102D of the sacrificial layer 102C. As shown in fig. 10A, when the etching holes 102D are, for example, cylindrical, the hydrophobic liquid 111 enters first, and this space becomes a fluid-dynamically immobile region, and therefore, when the droplet 110 is transferred to the etching holes 102D, the hydrophobic liquid 111 remains in the etching holes 102D without being rapidly replaced by a fluid. Such a phenomenon is easily generated in the hydrophobic liquid which is often used in EWOD. That is, since the hydrophobic liquid has a chemical property of low viscosity and low surface tension, if it has a structure having an immobile region like the cylindrical etching holes 102D, a phenomenon occurs in which replacement is impossible. In particular, when the density of the hydrophobic liquid 111 is higher than that of the liquid droplets, the buoyancy acts in opposition to the displacement, and therefore the displacement becomes more difficult.

Therefore, a structure for preventing the hydrophobic liquid 111 from remaining in the etching holes 102D of the sacrificial layer 102C will be described below.

Fig. 10B is a schematic diagram showing the configuration of the sacrificial layer 102C of this embodiment. As shown in fig. 10B, the sacrificial layer 102C of the present embodiment is formed such that the cross-sectional shape of the etching hole 102D (recess) is tapered. By providing the etching holes 102D with a structure having no fluid immobile region such as a tapered cross-sectional shape, the hydrophobic liquid 111 can be easily replaced fluidically by droplets of the electrolyte solution.

In the case where the etching holes 102D have a cylindrical shape, the electrolyte solution is sealed in the cylindrical etching holes 102D before the first liquid tank 104A is filled with the hydrophobic liquid 111, whereby the hydrophobic liquid 111 can be prevented from remaining. Since the liquid inside the cylindrical etching holes 102D is difficult to be replaced in a fluid manner, in the case where the hydrophobic liquid 111 is moved over later, the hydrophobic liquid 111 does not enter the etching holes 102D. In this case, by using a fluid having a lower specific gravity than water as the hydrophobic liquid 111, it becomes more difficult for the hydrophobic liquid 111 to enter into the etching holes 102D.

Fig. 10C is a schematic diagram showing another biopolymer analysis device 900 according to the present embodiment. As shown in fig. 10C, the structure of the thin film 102A, the tapered layer 102B, and the sacrificial layer 102C of the biopolymer analysis device 900 is the same as that of the biopolymer analysis device 500 (fig. 5) of embodiment 1, but the substrate 113 provided with the plurality of individual electrodes 112 is disposed in the 2 nd liquid bath 104B, and the common electrode 105 is disposed in the 1 st liquid bath 104A. Further, the plurality of droplets 110 and the hydrophobic liquid 111 are introduced into the 2 nd liquid tank 104B, and the electrolyte solution 103 is introduced into the 1 st liquid tank 104A.

Thus, the hydrophobic liquid 111 is filled on the tapered layer 102B side (the 2 nd liquid tank 104B), and then the liquid droplet 110 is transported, whereby the hydrophobic liquid 111 can be easily replaced with the liquid droplet 110 in a fluid manner.

< technical effects >

As described above, in the present embodiment, the etching hole 102D formed in the sacrificial layer 102C has a tapered cross-sectional shape. Alternatively, the cylindrical etching holes 102D are filled with the electrolytic solution in advance. Further, a plurality of individual electrodes 112 may be provided on the tapered layer 102B side (the 2 nd liquid bath 104B), and the hydrophobic liquid 111 and the liquid droplets 110 may be introduced. This prevents the hydrophobic liquid 111 from remaining in the etching holes 102D formed in the sacrificial layer 102C and causing a defective channel.

[ 5 th embodiment ]

< example of configuration of biopolymer analysis device >

Fig. 11 is a schematic diagram showing a biopolymer analysis device 1000 according to embodiment 5. As shown in fig. 11, a biopolymer analysis device 1000 according to this embodiment is different from those according to embodiment 1 (fig. 4) and embodiment 2 (fig. 7) in that an EWOD electrode 114 is formed on the upper surface of a sacrificial layer 102C (thin film). An insulator 115 is disposed on the surface of the EWOD electrode 114. Each EWOD electrode 114 is connected to an external circuit via a wiring (not shown) provided inside the sacrifice layer 102C. The droplets 110 are transported to positions where they are in contact with one individual electrode 112 and with at least two adjacent electrodes 114 for EWOD, respectively.

Fig. 12 is a schematic diagram showing another biopolymer analysis device 1100 according to embodiment 5. As shown in fig. 11, a biopolymer analysis device 1100 according to this embodiment differs from embodiments 1 (fig. 4) and 2 (fig. 7) in that a plurality of individual electrodes 112 (a plurality of 3 rd electrodes) for current measurement are formed on the upper surface of a sacrificial layer 102C (thin film), and only an EWOD electrode 114 (a plurality of 1 st electrodes) is formed on a substrate 113. Each individual electrode 112 is connected to an external circuit via a wiring (not shown) provided inside the sacrificial layer 102C. The droplets 110 are transported to positions where they are in contact with one individual electrode 112 and with at least two adjacent electrodes 114 for EWOD, respectively. In other words, the individual electrodes 112 are respectively configured to be in contact with one droplet 110.

< technical effects >

As described above, the biopolymer analysis devices 1000 and 1100 according to the present embodiment have the individual electrode 112 for current measurement and the electrode 114 for EWOD, and are configured such that either one of the individual electrode 112 or the electrode 114 for EWOD is integrated with the sacrificial layer 102C on the thin film 102A. As a result, compared to the case where both the individual electrode 112 for current measurement and the electrode 114 for EWOD are provided on the substrate 113 as in embodiment 2, the channels can be further integrated, and measurement of droplets using a smaller volume can be performed.

[ 6 th embodiment ]

In embodiment 1, as shown in fig. 3A, a configuration in which a substrate 113 having a plurality of individual electrodes 112 is disposed on one side of a thin film 102 (1 st liquid tank 104A) and droplets 110 are introduced has been described. On the other hand, in embodiment 6, a substrate 113 having a plurality of individual electrodes 112 is disposed on both sides of the thin film 102 (the 1 st liquid tank 104A and the 2 nd liquid tank 104B), and droplets 110 are introduced respectively.

< example of configuration of biopolymer analysis device >

Fig. 13 is a schematic diagram showing a biopolymer analysis device 1200 according to embodiment 6. As shown in fig. 13, a biopolymer analysis device 1200 according to the present embodiment includes a thin film 102, a1 st liquid tank 104A, a 2 nd liquid tank 104B, a substrate 113A having a plurality of individual electrodes 112A (a plurality of 1 st electrodes), and a substrate 113B having a plurality of individual electrodes 112B (a plurality of 2 nd electrodes). The substrate 113A is disposed in the 1 st liquid bath 104A, and the substrate 113B is disposed in the 2 nd liquid bath 104B. The plurality of individual electrodes 112A and the plurality of individual electrodes 112B are disposed at positions facing each other with the thin film 102 interposed therebetween.

A plurality of droplets 110 (measurement solution) and a hydrophobic liquid 111 are introduced into the 1 st liquid tank 104A and the 2 nd liquid tank 104B, respectively. Each droplet 110 is electrically insulated from adjacent droplets 110 by a hydrophobic liquid 111, independent of each other. Further, the plurality of droplets 110 are in contact with the individual electrodes 112, respectively, whereby electric operations such as application of voltage can be performed on the droplets 110. The other structures are the same as those of the biopolymer analysis device 300 (fig. 3) of embodiment 1, and therefore, the description thereof is omitted.

< method for analyzing biopolymer >

Since the method for analyzing a biopolymer according to the present embodiment is substantially the same as that of embodiment 1, the method for analyzing a biopolymer according to the present embodiment will be described with reference to fig. 6. Note that the same steps as those in embodiment 1 will not be described.

First, steps S1 to S6 of embodiment 1 are performed, and the hydrophobic liquid 111 and the droplets 110 are introduced into the 1 st liquid tank 104A to form a plurality of individual solution tanks. Then, in place of step S7, the hydrophobic liquid 111 and the droplets 110 are introduced into the second liquid tank 104B in the same manner as in steps S1 to S6 to form a plurality of individual solution tanks.

Next, in step S8, computer 108 applies a voltage equal to or higher than the dielectric breakdown voltage of film 102 between individual electrode 112A and individual electrode 112B facing each other, and electrically opens nanopore 101.

In steps S9 and S10, the voltage for EWOD transport is applied to the individual electrode 112A, the droplet 110 for nanopore boring is discarded from the 1 st liquid tank 104A, a sample solution is introduced into the 1 st liquid tank 104A, and sample measurement is performed, and then the voltage for EWOD transport is applied to the individual electrode 112B in the same manner in the 2 nd liquid tank 104B, and the droplet 110 for nanopore boring is replaced with the sample solution. Then, by inverting the voltage applied between the opposing individual electrode 112A and individual electrode 112B, sample measurement can be performed on the sample solution introduced into the 2 nd liquid bath 104B.

< technical effects >

As described above, in the present embodiment, the substrate 113 having the plurality of individual electrodes 112 is provided in both the 1 st liquid tank 104A and the 2 nd liquid tank 104B, and the droplets 110 are transported by EWOD. Thus, compared to embodiment 1 in which the sample solution is introduced into only one liquid tank (1 st liquid tank 104A), the number of samples can be measured twice without replacing the sample solution.

[ 7 th embodiment ]

In embodiment 1, the structure in which the 1 st liquid tank 104A is one layer is described, but the inside of the 1 st liquid tank 104A may have a two-layer structure of a layer for transporting the liquid droplets 110 and a layer for measuring a sample.

< example of configuration of biopolymer analysis device >

Fig. 14A is a schematic diagram showing a biopolymer analysis device 1300 according to embodiment 7. As shown in fig. 14A, in the biopolymer analysis device 1300 of the present embodiment, the substrate 113 constituting the upper surface of the 1 st liquid tank 104A is disposed, the substrate 119 is disposed in the 1 st liquid tank 104A substantially parallel to the substrate 113, and the 1 st liquid tank 104A has a two-layer structure. A plurality of EWOD electrodes 114 (a plurality of 1 st electrodes) are provided on the substrate 113, and the plurality of EWOD electrodes 114 are covered with insulators 115, respectively. The substrate 119 is provided with a plurality of individual electrodes 112 (a plurality of 3 rd electrodes) and a plurality of openings 120 through which the droplets 110 transported between the substrate 113 and the substrate 119 can pass.

After the first liquid tank 104A is filled with the hydrophobic liquid 111, a plurality of droplets 110 are introduced into an upper layer (between the substrate 113 and the substrate 119) of the first liquid tank 104A, and an EWOD transport voltage is applied between adjacent EWOD electrodes 114, whereby the droplets 110 are transported. When each droplet 110 is transported to the position of the opening 120, the droplet 110 moves to the lower layer (between the substrate 119 and the thin film 102) through the opening 120. The droplets 110 can move from the upper layer to the lower layer of the 1 st liquid tank 104A by gravity, buoyancy, or a difference in surface tension of the substrate surface with respect to water.

The substrate 119 may be subjected to hydrophilization treatment on the wall surface of the opening 120. Thereby, the droplet 110 can be more easily moved to the lower layer.

Fig. 14B is a schematic diagram showing a state in which a plurality of droplets 110 are arranged in the lower layer of the 1 st liquid tank 104A. As shown in fig. 14B, each individual electrode 112 is configured to contact one droplet 110 when each droplet 110 moves to the lower layer through the opening 120. Thus, an individual solution tank in which one individual electrode 112 is in contact with one droplet 110 is formed, and by applying an insulation breakdown voltage or an amperometric measurement voltage between the individual electrode 112 and the common electrode 105, the nanopore opening of the thin film 102 and the measurement of the sample can be performed.

< 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 disposed in the 1 st liquid tank 104A. Thus, the plurality of EWOD electrodes 114 and the plurality of individual electrodes 112 can be arranged at higher density on the substrates 113 and 119, as compared with embodiment 2 in which the plurality of EWOD electrodes 114 and the plurality of individual electrodes 112 are provided on the substrate 113.

[ 8 th embodiment ]

In embodiments 1 to 7, the configuration of a biopolymer analysis device has been mainly described. In the following, a biopolymer analysis device using a biopolymer analysis device will be described in this embodiment. As the biopolymer analysis device provided in the biopolymer analysis device, any one of the biopolymer analysis devices of embodiments 1 to 7 may be used.

< example of configuration of biopolymer analysis device >

Fig. 15 is a schematic diagram showing a configuration example of the biopolymer analysis device 1800. The biopolymer analysis device 1800 includes, as an example, the biopolymer analysis device 700 (see fig. 7) according to embodiment 2, a control circuit 121, and a computer 108 (control unit).

As shown in fig. 15, a plurality of droplets 110 (sample solution) containing the biopolymer 1 are transported in the 1 st liquid bath 104A, and no nanopore is formed in the thin film 102A. The electrolyte solution 103 is introduced into the 2 nd liquid tank 104B. In this way, the liquid droplets 110 containing the biopolymer 1 are used to form nanopores in the thin film 102A, and the biopolymer 1 can be directly analyzed. In this case, since the solution for opening the nanopore and the sample solution do not need to be replaced, the measurement time can be shortened.

Although not shown, a circuit for EWOD conveyance, a circuit for nanopore drilling, a circuit for ammeter measurement, and a switch for switching these circuits are provided inside the control circuit 121. The individual electrodes 112 and the common electrode 105 are connected to the nanopore opening circuit and the current measurement circuit via wires. The EWOD electrode 114 is connected to an EWOD transmission circuit via a wiring.

The ammeter measurement circuit is provided with an ammeter for measuring an ion current (blocking current) flowing between each individual electrode 112 and the common electrode 105. The ammeter has an amplifier and an analog/digital converter that amplify the current flowing between the individual electrode 112 and the common electrode 105. The ammeter is connected to the computer 108, and the analog/digital converter outputs the value of the detected ion current to the computer 108 as a digital signal.

The computer 108 is a terminal such as a personal computer, a smartphone, or a tablet, and has a data processing unit that processes various data, and a storage unit that stores an output value of an ammeter, data calculated by the data processing unit, and the like. The data processing unit counts the biopolymer 1 or acquires the monomer sequence information of the biopolymer 1 based on the current value of the ion current (blocking current) output from the ammeter. The data processing unit determines the position of the droplet 110, whether or not a leak is generated between the droplets 110, and whether or not a nanopore is formed in the thin film 102, based on the electrical characteristics such as the measured current value.

The computer 108 controls switching of the switches of the control circuit 121 and application of voltages to the common electrode 105, the individual electrodes 112, and the EWOD electrodes 114.

As shown in fig. 15, the control circuit 121 and the computer 108 may be integrated with the biopolymer analysis device instead of being separate components from the biopolymer analysis device 700.

< analysis of biopolymer >

In the state shown in fig. 15, when a voltage for forming a nanopore is applied between each individual electrode 112 and the common electrode 105, a nanopore is formed in the thin film 102A. Then, when a voltage for current measurement is applied between the individual electrode 112 and the common electrode 105, a potential difference is generated between both surfaces of the film 102A, and the biopolymer 1 dissolved in the liquid droplets 110 is electrophoresed in the direction of the common electrode 105. When the biopolymer 1 is DNA, since the droplets 110 are negatively charged, the biopolymer 1 can be electrophoresed in the direction of the common electrode 105 by setting the common electrode 105 to be a positive electrode. If the biopolymer 1 passes through the nanopore, a blocking current flows.

In the blockade current measurement using the biopolymer analysis device, the decrease in current observed when the nanopore is sealed with the biopolymer 1 (the nanopore is blocked by the biopolymer 1) is measured with the current value measured in the absence of the biopolymer 1 as a reference (pore current), and the speed and state of passage of the molecule are observed. When the biopolymer 1 passes through the nanopore, the acquired current value returns to the pore current. The nanopore passage speed of the biopolymer 1 can be analyzed from the blocking time, and the properties of the biopolymer 1 can be analyzed from the amount of blocking.

In the nanopore method for analyzing a biopolymer by an electric signal, particularly a signal change of an ionic current, the higher the conductivity of an electrolyte solution, the larger the signal change amount of the ionic current, and therefore, measurement at a high SN ratio is possible. The conductivity of the electrolyte solution can be improved by increasing the ionic strength, i.e., the salt concentration, although it depends on the transport number of the ion species, etc. Therefore, in nanopore analysis, measurement is performed at a salt concentration as high as possible from the viewpoint of the SN ratio. In particular, in nanopore analysis, a potassium chloride aqueous solution having a concentration of 1M is often used, and in some cases, a high salt concentration condition having an ionic strength of 3M or more is used. The maximum salt concentration is the upper limit at which the electrolyte can be dissolved, i.e., the saturation concentration.

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 3M can be used as the droplet 110 and the electrolyte solution 103. This is because the chloride ions can undergo an electron transfer reaction with the silver/silver chloride electrode, and the electric conductivities of the potassium ions and the chloride ions are equal to each other, so that the conductivity can be sufficiently ensured. The ion species may be, in addition to potassium chloride, lithium ions, sodium ions, rubidium ions, cesium ions, ammonium ions, or the like, which are monovalent cations of alkali metals.

< control of biopolymer transport >

When DNA sequencing or RNA sequencing is performed using the biopolymer analysis device 1800, it is necessary to control the transport of DNA or RNA when it passes through a nanopore. The biopolymer can be controlled mainly by a molecular motor using an enzyme. Molecular motor based delivery control needs to start only near the nanopore. In particular, by binding a control chain to a biopolymer to be read, the start of transport by a molecular motor in the vicinity of a nanopore can be controlled. Such structures are described in Japanese patent application No. 2018-159481 and PCT/JP2018/039466, for example. The disclosures of these documents are cited as constituting a part of this specification.

The enzyme used as the molecular motor herein refers to all enzymes having a binding ability to a biopolymer. When the biopolymer is a DNA, examples thereof include a DNA polymerase (polymerase), a DNA helicase (helicase), an exonuclease (exouclase), and a DNA transposase (transposase). When the biopolymer is RNA, examples thereof include RNA polymerase, RNA helicase, RNA exonuclease, and RNA transposase.

As described above, when a voltage is applied to both ends of the nanopore placed in the electrolyte solution, an electric field is generated in the vicinity of the nanopore 101, and the force causes the biopolymer to pass through the nanopore. On the other hand, since the molecular motor is generally larger than the nanopore diameter, it cannot pass through the nanopore. In order to achieve this limitation, it is desirable that the nanopore diameter is in the range of 0.8nm, which is the lower limit value through which single-stranded DNA or single-stranded RNA can pass, to 3nm, which is the upper limit value through which enzymes as molecular motors do not pass. Under these conditions, the primers (primers) in the control strand approach molecular motors that reside near the nanopore, thereby initiating the elongation, dissociation reaction. As a result, the biopolymer is lifted up or lowered from the nanopore by the force generated when the molecular motor elongates or separates the complementary strand, and the biopolymer is analyzed based on the change in the ionic current obtained at that time.

Although the structure for acquiring the monomer sequence information of the biopolymer 1 based on the electric signal has been described above, the monomer sequence information of the biopolymer 1 can be acquired by a method for acquiring a tunnel current by providing an electrode inside a nanopore and a method for detecting a change in transistor characteristics. Further, the structure may be such that monomer sequence information of the biopolymer 1 is obtained based on the optical signal. That is, the method may be a method in which each monomer is labeled with a fluorescence wavelength having a characteristic property, and the sequence of each monomer is determined by measuring the fluorescence signal.

A biopolymer analysis device (nanopore device) for analyzing a biopolymer and a biopolymer analysis apparatus provided with the same include the above-described configuration as an element. The biopolymer analysis device and the biopolymer analysis apparatus can be provided together with instructions describing the procedure and the amount of use. Further, the biopolymer analysis device may be provided in a state in which it can be used immediately and in a state in which a nanopore is formed, or may be provided in a state in which it is formed at a providing destination.

[ modified examples ]

The present disclosure is not limited to the above-described embodiments, and includes various modifications. For example, the above-described embodiments are described in detail to explain the present disclosure easily and understandably, and all of the described configurations are not necessarily required. A part of one embodiment may be replaced with the structure of another embodiment. Note that the structure of another embodiment can be added to the structure of one embodiment. Further, a part of the structure of each embodiment can be added, deleted, or replaced with a part of the structure of another embodiment.

All publications and patent documents cited in this specification are herein considered to be incorporated by reference as if fully set forth.

Description of the reference numerals

1: a biopolymer;

101: a nanopore;

102: a film;

103: an electrolyte solution;

104A: a1 st liquid tank;

104B: a 2 nd liquid bath;

105: a common electrode;

106: an ammeter;

107: a power source;

108: a computer;

110: a droplet;

111: hydrophobic liquid;

112: a separate electrode;

113: a substrate;

114: an electrode for EWOD;

115: an insulator;

116: an EWOD transmission circuit;

117: a circuit for forming a nanopore;

118: a current measuring circuit;

119: a substrate;

120: an opening;

121: a control circuit;

122: a switch;

123: and a capacitor.