JP2020008493A - Device and method for analyzing biological sample - Google Patents

Abstract

To increase the degree of integration of a solid nanopore sequencer.SOLUTION: The biological sample analyzer includes: a membrane 101; a first layer 102 having a plurality of openings to expose the membrane 101, the first layer being deposited on one surface of the membrane; a second layer deposited on the first layer 102, the second layer having a plurality of dependent electrodes 104 provided to correspond to the openings of the first layer 102, respectively; a third layer 100 deposited on the other surface of the membrane, the third layer having a single opening so that the surface opposite to the region exposed by the openings of the first layer of one surface of the membrane is exposed; a common electrode 200 generating a potential difference among the dependent electrodes 104, the parts of the membrane 101 which are exposed by the openings on the first layer 101 being insulated from one another.SELECTED DRAWING: Figure 1

Description

  The present disclosure relates to a biological sample analyzer and a biological sample analysis method.

  BACKGROUND ART As a biological sample analyzer for analyzing biological samples such as DNA and protein, development of a nanopore sequencer of a closed current system has been advanced. The closed current type nanopore sequencer includes a thin film membrane having nanopores of the same size as a biological sample, and two liquid tanks having electrodes disposed above and below the membrane. The two tanks are filled with a solution, and a biological sample is introduced into one of the tanks. Then, a voltage is applied to the electrodes, and a change in a current value flowing between the electrodes when the biological sample passes through the nanopore is measured. The nanopore sequencer determines the structural characteristics of the biological sample from such measurements.

  There are two methods for forming a closed pore type nanopore sequencer, a "bio type" and a "solid type". The bio-based method is a method for forming a nanopore sequencer using biomolecules. In the bio-type, first, a membrane on which an amphipathic lipid bilayer is placed is placed in a liquid tank filled with a solution. Next, nanopores are formed by floating a modified protein (such as Mycobacterium Smegmatis Porin A) having nanometer-sized nanopores on the membrane of the lipid bilayer membrane. In addition, the bio-type method has a problem that a protein and a lipid bilayer membrane used are easily denatured.

  On the other hand, unlike the biomolecule, the solid type uses a material having high mechanical strength for the membrane, and thus can be a nanopore sequencer that is robust to the environment such as temperature, pH, and voltage. In the solid type, for example, a silicon nitride film is used as a membrane, and nanometer-sized nanopores are formed on the membrane by electron beam irradiation or voltage application. In a general solid-type nanopore sequencer, a material having high mechanical strength, such as a silicon nitride film, is formed on a substrate, and an opening is formed in the substrate to expose the silicon nitride film. Thereby, a substrate having a membrane is formed. Then, members constituting the liquid tank and the electrodes are attached to the upper and lower sides of the substrate having the membrane. The liquid tank is provided with at least two flow paths (for example, a flow path for introducing a solution and a flow path for discharging gas and excess liquid existing in the liquid tank).

  In the nanopore sequencer, in order to improve the reading throughput, it is important to arrange a plurality of nanopore sequencers in parallel and measure the structure of the biomolecule in parallel. In arraying a nanopore sequencer, it is necessary to parallelize not only the nanopore but also the peripheral structure such as an associated liquid tank and electrode.

  For example, Patent Literature 1 discloses an array of solid nanopore sequencers. In the nanopore sequencer described in Patent Literature 1, membranes are formed in an array on a silicon substrate, and a part to be a liquid tank and two flow paths are connected to each of the membranes arranged in the array to form two membranes. One of the channels is formed of an electrode material. That is, the nanopore sequencer described in Patent Literature 1 has a structure in which the footprint (or installation area) of the channel and the electrode can be reduced by using the channel as the electrode.

JP 2012-26986 A

  By the way, in the nanopore sequencer described in Patent Document 1, the electrode provided independently in each sequencer is formed of a member different from the substrate having the membrane. Further, in the nanopore sequencer described in Patent Literature 1, two flow paths for inflow and outflow are provided to supply a solution to each of a plurality of independently provided liquid tanks. In the case of such a structure, the area of the nanopore sequencer increases due to the installation area of the flow path and another member. As a result, it becomes a factor that hinders integration and parallelization of the sequencer.

  The present disclosure has been made in view of the above points, and provides a technique for improving the integration degree of a solid nanopore sequencer.

  In order to solve the above problem, a membrane, a plurality of openings are provided so that the membrane is exposed, a first layer laminated on one surface of the membrane, and the first layer of the first layer A second layer laminated on the first layer, a second layer laminated on the first layer, and a second layer laminated on the other surface of the membrane; A third layer provided with a single opening such that a surface of the one surface opposite to a region exposed by the plurality of openings of the first layer is exposed; An electrode is a common electrode provided on the opposite side of the membrane, and is formed by a concave portion formed by the opening of the first layer and the single opening of the third layer. Conductive liquid is introduced into each of the recesses And a common electrode that generates a potential difference between the plurality of independent electrodes in a state where the plurality of independent electrodes are provided. Portions of the membrane that are exposed by the plurality of openings in the first layer are insulated from each other. A biological sample analyzer.

  A biological sample analysis method for simultaneously observing a plurality of changes in current when a sample passes through a nanopore, wherein a plurality of openings provided in a first layer laminated on a membrane on which the nanopore is formed are provided. For the plurality of recesses formed by the surface of the membrane, a step of introducing a solution from an inlet provided in a liquid tank support surrounding the plurality of openings and filling the same with the solution; An insoluble and insulative liquid for the solution is introduced from the inlet, and a part of the solution is caused to flow out of an outlet provided in the liquid tank support. A step of forming droplet-shaped shapes that do not come into contact with each other; a step of introducing a solution into an opening provided in a substrate supporting the membrane; and a step of sandwiching the membrane. Forming nanopores in a plurality of partial regions of the membrane exposed by the opening by applying a voltage between a plurality of independent electrodes and a single common electrode; and introducing the nanopores into the plurality of recesses. Measuring a current value flowing through the nanopore when a biological sample contained in one of the solution and the solution introduced into the opening provided in the substrate passes through the nanopore. Provided is a biological sample analysis method.

  According to the present disclosure, the integration degree of a solid nanopore sequencer can be improved. Further features related to the present disclosure will become apparent from the description of the present specification and the accompanying drawings. Problems, configurations, and effects other than those described above will be clarified by the following description of the embodiments.

FIG. 2 is a cross-sectional view of an analysis device included in the biological sample analyzer according to the first embodiment. FIG. 1 is a diagram schematically illustrating an overall configuration of a biological sample analyzer according to a first embodiment. 6 is a flowchart showing a flow of analyzing a biological sample using the biological sample analyzer. FIG. 3 is a diagram illustrating a state in which a solution supply mechanism introduces various solutions into an analysis device. FIG. 3 is a diagram illustrating a state in which a solution supply mechanism introduces various solutions into an analysis device. FIG. 3 is a diagram illustrating a state in which a solution supply mechanism introduces various solutions into an analysis device. FIG. 3 is a diagram illustrating a state in which a solution supply mechanism introduces various solutions into an analysis device. It is a figure showing signs that a biological sample was introduced into a common liquid tank. FIG. 7 is a top view of the analysis device when the number of arrays is 64 (8 rows, 8 columns). FIG. 9 is a diagram for explaining a method for introducing a solution in Example 2. It is a top view of the analysis device in the case where the number of arrays is 256 (arrays of (8 rows, 8 columns) are arranged in (2 rows, 2 columns)). FIG. 9 is a partial cross-sectional view of the analysis device according to the third embodiment. It is a figure showing signs that board opening 108A and board opening 108B are divided by a support part. FIG. 14 is a cross-sectional view of the analysis device according to the fourth embodiment. It is a figure showing another example of a solution support.

  Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. Each drawing is schematically drawn, and portions unnecessary for description are omitted. The structures, materials, and methods described in the embodiments are examples for embodying the idea of the present disclosure, and do not strictly specify materials, dimensions, and the like. The embodiments of the present disclosure are not limited to the embodiments described below, and various modifications are possible within the scope of the technical idea.

<Example 1>
FIG. 1 is a cross-sectional view of an analysis device 10 provided in the biological sample analyzer according to the first embodiment. The biological sample analyzer includes a substrate 100 (third layer), a membrane 101 (101A, 101B, 101C, 101D), an insulating layer 102 (first layer), a wiring 103 (103A, 103B, 103C, 103D), An electrode 104 (104A, 104B, 104C, 104D), an I / O pad 105 (105A, 105B, 105C, 105D), a partition 106, a recess 107 (107A, 107B, 107C, 107D) and a common electrode 200 are provided. A substrate opening 108 is provided in the substrate 100, and the substrate opening 108 functions as the common liquid tank 201.

  An insulating layer 102 (first layer) is stacked on one surface of the membrane 101, and a substrate 100 (third layer) is stacked on the other surface. A plurality of openings are provided in the insulating layer 102 so that the membrane 101 is exposed, and a plurality of independent electrodes 104 are provided in an array in association with each of the plurality of openings. That is, the plurality of independent electrodes 104 are provided above the membrane 101, and the membrane 101 is exposed at the bottom of the concave portion 107 formed by the insulating layer 102 and the partition 106. Note that one independent electrode 104, one recess 107, and the partition 106 surrounding the recess 107 define one section. That is, the biological sample analyzer can simultaneously execute a plurality of biological sample analyzes by the number of sections.

  In addition, each of the plurality of independent electrodes 104 is connected to the I / O pad 105 via a wiring 103 patterned while being covered with the insulating layer 102. The partition 106 has one of a plurality of openings (for example, a first layer portion of the concave portion 107A) of the insulating layer 102 and one of a plurality of independent electrodes 104 associated with one of the plurality of openings ( For example, a pair of independent electrodes 104A), a plurality of independent electrodes associated with another one of the plurality of openings (for example, the first layer portion of the recess 107B) and another one of the plurality of openings. Another pair of the electrodes 104 (for example, independent electrodes 104B) are provided so as to be isolated from each other.

  On the other hand, the substrate opening 108 and the common electrode 200 are arranged on the opposite side to the plurality of independent electrodes 104 with the membrane 101 interposed therebetween. The common liquid tank 201 forms one space connected to the substrate opening 108.

  Here, for example, the material of the substrate 100 is silicon, the material of the membrane 101 is silicon nitride, the material of the insulating layer 102 is silicon oxide, the material of the wiring 103 is copper, the independent electrode 104, the common electrode The material of the I / O pad 200 and the I / O pad is gold, the material of the partition 106 is a polyimide resin, and the material of the common liquid tank 201 is an acrylic resin.

  The surface of the membrane 101 is hydrophilic, and the upper surface and side surfaces of the partition 106 surrounding the membrane 101 are hydrophobic. Here, as an example, the diameter of the membrane 101 was 200 nm, the short dimension of the first electrode 104 was 1 μm, the opening diameter of the partition 106 was 25 μm, and the array pitch was 50 μm. Note that the partition wall 106 is not necessarily hydrophobic or is not sufficiently hydrophobic as long as, for example, the surface is subjected to a hydrophobic treatment. In this case, the hydrophobic treatment may be performed only on the upper surface of the partition 106, or may be performed over the entire surface of the partition 106.

  The analysis device 1 can be formed, for example, by a method similar to a semiconductor process. The wiring pattern is formed by, for example, a damascene method. Specifically, after forming the layer, a resist is applied, etched along a desired pattern, and a wiring pattern is formed by electrolytic plating on the etched portion. After that, unnecessary resist is removed by ashing, and the surface is flattened by CMP (Chemical Mechanical Polishing). By repeating this process, a necessary wiring pattern, independent electrodes 104, partition walls 106, and the like are obtained.

  FIG. 2 is a diagram schematically illustrating an overall configuration of the biological sample analyzer 1 according to the first embodiment. The biological sample analyzer 1 includes a control circuit unit 300, a power supply and control / detection data acquisition unit 400, and a solution supply mechanism 500. The control circuit unit 300 is connected to the I / O pad 105 by a detachable terminal, and is connected to the power supply and the control / detection data acquisition unit 400 via wiring. The control circuit unit 300 controls, for example, a voltage applied to the plurality of independent electrodes 104 and transfers a signal obtained during measurement to the power supply and the control / detection data acquisition unit 400 or a PC (not shown).

  The power supply and control / detection data acquisition unit 400 includes at least a high output power supply, a processor such as a CPU (Central Processing Unit), a memory, and a storage unit such as a hard disk. The memory stores, for example, a data analysis program and a device control program, and the CPU reads the program to measure measurement data and control various devices.

  The solution supply mechanism 500 is arranged above the analysis device (the structure shown in FIG. 1) and serves to supply a solution to the membrane 101. The solution supply mechanism 500 is controlled by, for example, a power supply and a control signal output from the control / detection data acquisition unit 400, and discharges a predetermined amount of liquid at a predetermined position. The solution supply mechanism 500 can also function as a suction mechanism by operating an actuator or a motor in a manner opposite to that at the time of discharging.

[Biological sample analysis flow]
FIG. 3 is a flowchart showing a flow of analyzing a biological sample using the biological sample analyzer 1. The case where the number of arrays is four will be described below, but the number of arrays may be more or less than four. Hereinafter, steps S11 to S21 will be described.

(1) Step S11
First, the solution 501 and the biological sample 502 are prepared in the solution supply mechanism 500, and the solution supply mechanism 500 is moved to a desired position under the control of the power supply and the control / detection data acquisition unit 400. Then, the solution supply mechanism 500 introduces the solution 501 containing the biological sample 502 into the concave portion 107 having the membrane 101 at the bottom.

  FIG. 4 is a diagram showing how the solution supply mechanism 500 introduces various solutions into the analysis device 10. As shown in FIG. 4A, when supplying the solution 501 to one concave portion 107A, first, the solution supply mechanism 500 is aligned with the concave portion 107A, and then the concave portion 107A is filled (the membrane 101A and the independent electrode are filled). A sufficient amount of the solution 501 is introduced into the adjacent concave portion 107B, which is enough to immerse the solution 501 in the solution. This step was also performed on other concave portions (107B, 107C, and 107D), and all the concave portions 107 were filled with the solution 501 as shown in FIG. Here, the solutions 501 introduced between the adjacent concave portions 107 form parallel liquid pools (503A, 503B, 503C, 503D) that are blocked so as not to contact each other. Note that the solution 501 contains an electrolyte such as potassium chloride. Further, the solution supply mechanism 500 may include a plurality of projecting ports, and may introduce the solution in parallel to each of the recesses (107A, 107B, 107C, 107D).

(2) Step S12
Next, the insulating fluid 504 is prepared in the solution supply mechanism 500. As shown in FIG. 4C, the insulating fluid 504 is introduced from above the parallel liquid reservoirs (503A, 503B, 503C, 503D) from the solution supply mechanism 500, and the parallel liquid reservoirs (503A, 503B, 503C) are introduced. , 503D). The presence of the insulating fluid 504 prevents a minute amount of liquid pool (503A, 503B, 503C, 503D) from drying. Here, the insulating fluid 504 is, for example, an oily liquid and is insoluble in the solution 501.

(3) Step S13
Subsequently, the solution 501 is introduced into the common liquid tank 201 as shown in FIG. Thereby, the upper and lower surfaces of the membrane 101 are filled with the solution. Note that the order of the set of steps S11 and S12 and step S13 may be reversed. That is, the steps may be performed in the order of step S13, step S11, and step S12.

(4) Step S14
A voltage is applied between adjacent independent electrodes (104A, 104B, 104C, 104D), and a leak current value is measured. For example, the leak current value between the independent electrode 104A and the independent electrode 104B and the leak current value between the independent electrode 104C and the independent electrode 104D are measured in parallel, and then the leak current value between the independent electrode 104B and the independent electrode 104C is measured. Is measured. This is because if there is a leak of the solution 501 (that is, contact between droplets) between the independent electrodes (104A, 104B, 104C, 104D), no nanopores are formed on the membrane 101 in the nanopore formation step described later, or This is to identify a defective section in which a leak has occurred because various problems occur such as generation of signal noise in the sample analysis step. Step S14 may be performed after step S11. For example, step S14 may be performed before step S13.

(5) Step S15
To determine whether or not there is a leak current, a constant current value that does not hinder the nanopore formation step and the sample analysis step described below is set as a threshold, and the measured leak current value is compared with the threshold. For example, when the applied voltage is 0.1 V, the threshold is set to 10 pA, which is sufficiently smaller than the current flowing through the nanopore in step S21 described below. If it is determined in step S15 that the leak current value is equal to or larger than the threshold, the process proceeds to step S22, and the section in which the leak current is detected is identified as defective. The defective section or the non-defective section is stored in a storage unit (for example, a memory included in a power supply and a control / detection data acquisition unit). If the leak current value is smaller than the threshold value in the determination in step S15, the process proceeds to step S16, and a section in which no leak current is detected is determined as a good section.

(6) Step S16
A voltage lower than that applied in step S18 is applied between the common electrode 200 and each of the independent electrodes (104A, 104B, 104C, 104D), and flows through each of the membranes (101A, 101B, 101C, 101D). Measure the leak current between the membranes. At this time, the applied voltage is, for example, a sweep voltage in a range of −1 to +1 V.

(7) Step S17
For the inter-membrane leak current, a certain level that does not cause any trouble in the sample analysis process described later is set as a threshold, and the measured inter-membrane leak current is compared with the threshold. For example, when there is an initial failure such as a previously cracked membrane, a large current flows between the membranes. Therefore, for example, when the voltage applied between the membranes is 0.1 V, the threshold value is set to 100 pA, which is sufficiently smaller than the current flowing through the nanopore in step S21 described below. If the inter-membrane leak current value is equal to or greater than the threshold value in the determination of step S17, the process proceeds to step S22, and the section in which the leak current is detected is identified as defective. The defective section or the non-defective section (the section not determined to be defective) is stored in the storage section. If the inter-membrane leakage current value is smaller than the threshold value in step S17, the process proceeds to step S18, and the section in which no leak current is detected is determined as a non-defective section.

(8) Step S18
A voltage is applied between the common electrode 200 and each of the independent electrodes (104A, 104B, 104C, 104D), and a nanometer-sized membrane is applied to the membrane (101A, 101B, 101C, 101D) by a known dielectric breakdown mechanism. Form nanopores.

(9) Step S19
In order to determine whether a nanopore of a desired size has been formed, a voltage smaller than the voltage applied in the nanopore formation step is applied between the common electrode 200 and the independent electrodes (104A, 104B, 104C, 104D), and the common electrode is formed. The value of the current flowing between 200 and each of the independent electrodes (104A, 104B, 104C, 104D) is measured. The diameter of the formed nanopores increases as the value of the current flowing through the membrane 101 increases, and can be controlled by the current value (see WO2015 / 099775A). The process in step S19 is performed when the membrane 101 has insufficient hydrophilicity and the membrane 101 does not sufficiently contact the solution 501 because a voltage is not normally applied above and below the membrane 101 and nanopores are not formed. It is.

(10) Step S20
It is determined whether the value of the current flowing between the common electrode 200 and each of the independent electrodes (104A, 104B, 104C, 104D) is a value corresponding to a nanopore of a desired size. If there is a section in which the current value flowing between the common electrode 200 and each of the independent electrodes (104A, 104B, 104C, 104D) is smaller than a predetermined current value, the section is determined to be defective. Thereafter, the process proceeds to step S22, and the defective section is stored in the storage unit.

  The voltage and sequence applied to each independent electrode 104 are controlled by the control circuit unit 300, and nanopores are formed in a plurality of sections (a plurality of exposed portions of the membrane 101) in parallel. At this time, it is not necessary to perform the step of applying the voltage and the step of measuring the current to the section determined to be defective after the formation of the liquid tank described above. If the nanopore formation step and the current value measurement step are performed only on the non-defective sections, the nanopore formation can be performed efficiently. However, if this defect occurs due to air bubbles between the membrane 101 and the solution 501, the work may be relieved by removing the air bubbles. If there are air bubbles between the membrane 101 and the solution 501, the analysis device 10 is placed under a reduced pressure environment to expand the volume of the air bubbles, increase the buoyancy of the air bubbles, and remove the air bubbles. It returns to S18.

(11) Step S21
Finally, a voltage is applied between the common electrode 200 and each of the independent electrodes (104A, 104B, 104C, 104D). By applying a voltage, an electric field is generated around the nanopore, and electrophoresis of the charged biological sample 502 in the liquid occurs. Thereby, the biological sample 502 is introduced into each nanopore, and passes through the nanopore. The detected current value differs before the biological sample passes through the nanopore and when the biological sample passes through the nanopore. This is because the nanopore is partially blocked by the cross-sectional area of the biological sample, and the resistance value of the nanopore changes. The structure of the biological sample is analyzed from the measured current values. Note that the structural analysis of the sample by the measurement of the blocking current may be performed on a section that is not a defective section.

  Through the above steps, a highly integrated structure in which parallel elements such as the membrane 101, the independent electrode 104, and the concave portion 107 are formed on the substrate 100 without a flow path is realized, and parallel nanopore formation and sample analysis are efficiently performed. Is realized.

  The above description is an example, and the biological sample analyzer 1 is not limited to the above configuration. If the alignment with the substrate 100 and the adjustment of the solution discharge amount do not cause a problem, the solution supply mechanism 500 does not need to be incorporated in the apparatus, and the solution discharge operation may be performed by hand using a micropipette.

  In the above description, the control circuit unit 300 is an independent component. However, the control circuit unit 300 may be provided on the board 100, or may be arranged in the power supply and the control / detection data acquisition unit 400. is there. That is, the biological sample analyzer 1 may be constructed as a system suitable for the measurement environment.

  Further, different types of solutions 501 and biological samples 502 can be introduced into each of the plurality of independent concave portions 107. In that case, it is not necessary to change the type of the solution 501 and the biological sample 502 in each of the plurality of recesses (107A, 107B, 107C, 107D), and the combination is arbitrary. Accordingly, if the same sample 502 is mixed into the same solution 501 and a plurality of analyzes are performed, analysis accuracy can be improved. If the same sample 502 is analyzed under different solution conditions, diversified information can be obtained. By arranging different samples 502, the measurement throughput can be improved.

When the same sample 502 is introduced into all the recesses 107 for analysis, the biological sample 502 may be introduced to the common liquid tank 201 side.
FIG. 5 is a diagram illustrating a state where the biological sample 502 is introduced into the common liquid tank 201. When the biological sample 502 is introduced into the common liquid tank 201, the measurement is performed, for example, by reversing the direction of the voltage applied to the independent electrode 104 and the common electrode 200, as compared with the case where the biological sample 502 is introduced into the recess 107. .

  Also, regarding the analysis device 10, if the membrane 101 and the independent electrode 104 are sufficiently hydrophilic and a hydrophobic region is formed so as to surround the membrane 101 and the independent electrode 104 in the same section, the partition 106 is not required. Is also good. In that case, for example, a hydrophobically-treated region is formed on the surface of the insulating layer 102 (first layer) so as to surround the exposed portion of the membrane 101 and the independent electrode 104 in the same section.

  In the case where the number of wirings 103 connecting the independent electrodes 104 and the I / O pads 105 is large in number and the wirings cannot be arranged with one layer, the layers provided with the wirings 103 may be multilayered. Further, for example, the material of the membrane 101 is silicon nitride, but may be silicon oxide, graphene, graphite, an organic substance, a polymer material, or the like. Although the material of the independent electrode 104 and the common electrode 200 is gold, other metals such as silver and platinum may be used. Although the material of the partition 106 is a polyimide resin, an insulating material such as an epoxy resin or silicon oxide may be used.

  Further, the substrate 100 is installed so that the membrane 101 is located above, but a configuration in which the substrate 100 is located above the membrane 101 may be employed. In this case, a solution 501 having a sufficient surface tension is selected so that a liquid tank is formed in the concave portion 107, and the solution 501 is sprayed from below toward the concave portion 107.

  FIG. 6 is a top view of the analysis device 10 when the number of arrays is 64 (8 rows, 8 columns). FIG. 6 illustrates a layout of the substrate 100, the membrane 101, the I / O pad 105, the partition 106, and the substrate opening 108. The partition 106 shown in FIG. 6 includes the membrane 101, the independent electrode 104, and the recess 107 shown in FIG. Further, the I / O pad 105 arranged around the substrate 100 is connected to the independent electrode 104 arranged around the exposed portion of the membrane 101 via the wiring 103 (not shown in FIG. 6) shown in FIG. It is connected. The substrate opening 108 formed on the back surface side of the substrate 100 has a surface opposite to a region exposed by the plurality of openings of the insulating layer 102 (first layer) on one surface of the membrane 101. It is provided so as to be exposed.

  Note that the nanopore may be formed on the membrane 101 in advance. In the biological sample analyzer 1 according to Example 1, the nanopore was formed by applying a voltage to the membrane. However, the method for forming the nanopore is not limited to this. The nanopore may be formed by previously irradiating the membrane 101 with an electron beam (for example, see A. J. Storm et al., Nat. Mat. 2 (2003)).

  As described above, when the solution 501 is introduced, the wettability between the membrane 101 and the solution 501 is high, and the solution 501 does not leak between different concave portions (for example, between the concave portions 107A and 107B) ( It is important that they do not touch each other. In order to enhance the wettability of the membrane 101, it is effective to perform a surface treatment before introducing the solution 501.

  As a surface treatment for improving wettability, the analysis device 10 may be immersed in a mixture of hydrogen sulfide and hydrogen peroxide to remove organic substances deposited on the surface of the membrane 101, and alcohol may be removed before the solution 501 is introduced. The solution may be introduced and replaced with the solution 501, and the analysis device 10 may be subjected to oxygen plasma treatment. Further, the above methods may be combined.

  When the surface treatment for improving the wettability enhances the wettability of the region between the two concave portions and causes leakage of the solution 501 between adjacent arrays, when performing the surface treatment for improving the wettability, It is effective to mask a region between the two concave portions. As a method of masking, there are a method of forming a photosensitive resist and then leaving the resist only in a mask region by using lithography, and a method of attaching a pre-patterned sheet. In the first embodiment, the partition wall 106 itself is made of a hydrophobic material, and the hydrophobicity is used for preventing leakage. However, if leakage in a region between the two concave portions becomes a problem, the surface of the partition wall 106 is positively hydrophobicized. Surface treatment to make the surface smooth. As a surface treatment for making the surface of the partition wall 106 hydrophobic, a fluorine plasma treatment may be performed, or a hydrophobic material such as hexamethyldisilazane may be formed on the surface. At this time, a hydrophobic surface treatment is applied to the entire surface of the array (the upper surface of the analysis device 10) in order to prevent the influence of the hydrophobic treatment on the region where the wettability should not be impaired, such as the exposed portion of the membrane 101. After that, the hydrophobically treated surface of the exposed portion of the membrane 101 may be removed, or a hydrophobic surface treatment may be performed in a state where the exposed portion of the membrane 101 is masked in advance, and the mask may be lifted off later. .

<Example 2>
In the second embodiment, an example of a method for introducing the solution 501 in which the throughput for introducing the solution 501 is improved as compared with the first embodiment will be described. Since the analysis method of the biological sample 502 described in the first embodiment is applied to the method other than the method of introducing the solution 501, the steps other than the method of introducing the solution 501 and the description of the structure of the biological sample analyzer 1 are omitted. That is, in the second embodiment, the content of the process of step S11 in the flowchart shown in FIG. 3 is different from that of the first embodiment. Therefore, here, only the process of step S11 in the case of the second embodiment will be described, and the description of the subsequent processes will be omitted.

  FIG. 7 is a diagram for explaining a method of introducing the solution 501 in the case of the second embodiment. As shown in FIG. 7, in the second embodiment, the introduction of the solution 501 into each of the concave portions 107 in step S11 is performed simultaneously on the concave portions 107A and the concave portions 107B by using the solution supply mechanism 500.

  In this case, first, the shape of the solution 501 is a shape that straddles the concave portions 107A and 107B. After that, the solution 501 is sucked by the solution supply mechanism 500. At least the independent electrodes 104A and 104B and the membranes 101A and 101B are sufficiently hydrophilic, and the partition 106 disposed between the membranes 101A and 101B has a hydrophobic surface. The solution 501 is divided and remains in a pool so that the solution 501 does not contact the concave portions 107A and 107B. When the solution 501 is sucked, for example, a motor or the like provided in the solution supply mechanism 500 is operated in the opposite direction to when discharging.

  By the above-described discharge operation and suction operation, the exposed portion of the membrane 101A and the independent electrode 104A in the concave portion 107A and the exposed portion of the membrane 101B and the independent electrode 104B in the concave portion 107B are filled with the solution 501. The solution 501 forms an independent parallel liquid pool between the adjacent concave portions 107A and 107B. The above process is also performed on the concave portions 107C and 107D to form a liquid pool on the entire array. When the method for introducing the solution 501 according to the second embodiment is applied, the same sample and the same solution can be simultaneously measured in the sections (for example, the concave portions 107A and the concave portions 107B) into which the solution 501 is collectively introduced. Not only can the solution be obtained, but also the solution 501 is simultaneously introduced into a plurality of compartments, so that the introduction throughput of the solution 501 is high.

<Example 3>
Hereinafter, a description will be given of a device structure in which the membrane 101 is less likely to be damaged and the defect rate is reduced as compared with the analysis device 10 according to the first embodiment. As shown in FIG. 4, the analysis device 10 according to the first embodiment has one substrate opening 108 for four recesses (107A, 107B, 107C, and 107D) and four recesses (107A). , 107B, 107C, 107D) are supported only by the substrate 100 at the outer periphery of the array. As shown in FIG. 6, in the analysis device 10 according to the first embodiment, the diameter of the substrate opening 108 is substantially the same as the diameter of the outer periphery of the partition 106. For this reason, when the number of arrays increases and the area of the substrate opening 108 increases, the portion of the membrane 101 exposed by the substrate opening 108 includes the stress of the insulating layer 102 and the partition 106 laminated thereon and the external foreign matter. It is easily damaged by contact with

  The biological sample analyzer according to the third embodiment differs from the biological sample analyzer 1 according to the first embodiment in the structure of the analysis device. Otherwise, the structure and method described in the first embodiment are applied, and description of the same steps and structures as in the first embodiment is omitted.

  FIG. 8 is a top view of the analysis device 20 when the number of arrays in the example is 256 (arrays of (8 rows, 8 columns) are arranged in (2 rows, 2 columns)). FIG. 8 illustrates a layout of the substrate 100, the membrane 101, the partition 106, and the substrate opening 108. FIG. 8 illustrates an example in which the analysis device 20 of the third embodiment has four substrate openings 108. Each of the four substrate openings 108 is arranged in a (2 rows, 2 columns) layout. ing. Each of the four substrate openings 108 includes a plurality of sections (or recesses 107).

  FIG. 9 is a cross-sectional view of a part of the analysis device 20 according to the third embodiment. In FIG. 9, the exposed portions 101A and 101B of the membrane are included in the same substrate opening 108A, and the exposed portions 101C and 101D of the membrane are included in the same substrate opening 108B. As shown in FIG. 9, there is a projection between the substrate opening 108A and the substrate opening 108B, and the strength of the substrate 100 is maintained. In addition, a support portion may be provided between the substrate opening 108A and the substrate opening 108B to form a plurality of common liquid tanks 201A and 201B.

  FIG. 10 is a diagram illustrating a state in which the substrate opening 108A and the substrate opening 108B are separated by the support portion S. For example, if a thermocompression bonding material processed by photolithography is used for the support portion S, the common liquid tanks 201A and 201B can be partitioned by a fine structure without increasing the device area. . However, the method of partitioning between the common liquid tanks 201A and 201B is not limited to this. Different solutions and / or different biological samples may be introduced into the common liquid tanks 201A and 201B partitioned by the support S. By doing so, the measurement throughput can be improved.

<Example 4>
In the biological sample analyzer 1 according to the first embodiment, the solution 501 and the biological sample 502 are introduced into the plurality of recesses 107 using the solution supply mechanism 500. In the biological sample analyzer according to the fourth embodiment, the solution 501 and the biological sample 502 are introduced into the analysis device by a method different from that in the first embodiment. The fourth embodiment is the same as the first embodiment except for the method of introducing the solution 501, and thus the description of the same steps and structures is omitted. Specifically, only the first steps S11 and S12 in the flowchart shown in FIG. 3 are different between the first embodiment and the fourth embodiment. Therefore, only the steps S11 and S12 in the fourth embodiment will be described.

  FIG. 11 is a cross-sectional view of the analysis device 30 according to the fourth embodiment. The analysis device 30 according to the fourth embodiment includes a liquid tank support 505 that covers the outer periphery of the partition wall 106. In the biological sample analyzer of the fourth embodiment, the solution 501 and the biological sample 502 are introduced into each of the plurality of recesses 107 by introducing the solution 501 and the biological sample 502 into the solution support 505 in step S11 in FIG.

  FIG. 11A is a diagram illustrating a state where the solution 501 is filled in the liquid tank support 505. In the case of the biological sample analyzer of the fourth embodiment, in step S11 in the flowchart of FIG. 3, the solution is introduced from the opening of the liquid tank support 505, and the exposed portions (101A, 101B, 101C, 101D) of the membrane are provided at the bottom. The solution is supplied to the concave portions (107A, 107B, 107C, 107D). In this step S11, the space between the exposed portions (101A, 101B, 101C, 101D) of the membrane is also filled with the solution 501.

  FIG. 11B is a diagram illustrating a state where an insulating fluid is introduced into the liquid tank support 505. In the case of the biological sample analyzer of the fourth embodiment, the insulating liquid 504 flows from the opening of the liquid tank support 505 in step S12. At least the independent electrodes (104A, 104B, 104C, 104D) and the exposed portions of the membrane (101A, 101B, 101C, 101D) are sufficiently hydrophilic, and are located between each of the plurality of recesses (107A, 107B, 107C, 107D). Of the partition 501 is a hydrophobic surface, the inflow of the insulating liquid 504 causes the solution 501 to be broken, and the liquid pools (503A, 503A, 503A, 107B, 107C, 107D) that do not contact each other. 503B, 503C, and 503D). In other words, the space between each of the exposed portions 101 of the membrane included in each of the plurality of recesses 107 and each of the plurality of independent electrodes 104 is filled with the solution 501, and the space between the adjacent recesses (for example, the recess 107A and the recess 107A). 107B), the solution is shut off. By using the above method, the solution 501 can be introduced into the plurality of recesses 107 at the same time, so that the introduction throughput of the solution 501 is high. Note that, when the insulating fluid is introduced, the solution 501 introduced into the concave portion 107 due to the insulating fluid overflowing from the discharge port through the upper portion of the liquid tank support 505 to prevent the solution 501 from being torn off. The bath support 505 needs to be designed at a sufficiently low height.

  FIG. 12 is a diagram illustrating another example of the solution support. As shown in FIG. 12, the liquid tank support 505 may be provided with a partition for dividing the array (the plurality of recesses 107). For the partition of the liquid tank support 505, for example, a thermocompression bonding material processed by photolithography is used. By doing so, the liquid tank support 505 can be partitioned by a fine structure, and the area of the analysis device does not increase. The method of partitioning the liquid tank support 505 is not limited to this. In the liquid tank support 505 having the above-mentioned partition, different solutions and / or different biological samples may be introduced into each of the plurality of partitioned liquid tank supports 505. In the analysis device shown in FIGS. 11 and 12, the solution supply mechanism 500 becomes unnecessary, and the cost of the apparatus can be reduced.

  As described above, the biological sample analyzer of the present disclosure eliminates a flow path and integrates a liquid tank and electrodes (independent electrodes and common electrodes) in a single analytical device when forming an array of solid nanopore sequencers. And parallelization. Therefore, the biological sample analyzer according to the present disclosure can not only make the device compact, for example, but also improve the measurement throughput.

  Note that the present disclosure is not limited to the above-described embodiments, and includes various modifications. For example, the above-described embodiments have been described in detail in order to easily explain the present disclosure, and are not necessarily limited to those having all the configurations described above. Further, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of one embodiment can be added to the configuration of another embodiment. Further, for a part of the configuration of each embodiment, it is possible to add / delete / replace another configuration.

100: substrate 101 (101A, 101B, 101C, 101D): membrane 102: insulating layer 103 (103A, 103B, 103C, 103D): wiring 104 (104A, 104B, 104C, 104D): independent electrode 105 (105A, 105B, 105C, 105D): I / O pad 106: partition 107 (107A, 107B, 107C, 107D): recess 108, 108A, 108B: substrate opening 200: second electrode 201, 201A, 201B: common liquid tank 300: Control circuit unit 400: power supply and control / detection data acquisition unit 500: solution supply mechanism 501: solution 502: biological sample 503 (503A, 503B, 503C, 503D): liquid pool 504: insulating fluid 505: liquid tank support

Claims (20)

With a membrane,
A plurality of openings are provided so that the membrane is exposed, and a first layer stacked on one surface of the membrane,
A second layer stacked on the first layer, the second layer having a plurality of independent electrodes provided in association with each of the plurality of openings in the first layer,
A single opening is stacked on the other surface of the membrane so that a surface of the one surface of the membrane opposite to a region exposed by the plurality of openings of the first layer is exposed. A third layer provided with a portion,
The independent electrode is a common electrode provided on the opposite side of the membrane, and includes a concave portion formed by the opening of the first layer, and the single opening of the third layer. A common electrode that generates a potential difference between the plurality of independent electrodes in a state where the conductive liquid is introduced into each of the recesses formed by
With
Portions of the membrane that are exposed by the plurality of openings in the first layer are insulated from each other,
Biological sample analyzer. The biological sample analyzer according to claim 1,
The surface of the first layer that is not in contact with the membrane is a hydrophobic surface surrounding one of the plurality of openings and one pair of the plurality of independent electrodes associated with one of the plurality of openings. Having a sex area,
Biological sample analyzer. The biological sample analyzer according to claim 1,
The second layer includes one of the plurality of openings included in the first layer, one pair of the plurality of independent electrodes associated with one of the plurality of openings, and the plurality of openings. Another pair of the plurality of independent electrodes associated with another one of the portions and another one of the plurality of openings, further comprising an insulating partition wall that isolates each other,
Biological sample analyzer. The biological sample analyzer according to claim 3,
The upper surface of the partition is hydrophobic,
Biological sample analyzer. The biological sample analyzer according to claim 4,
The side surface of the partition is hydrophobic,
Biological sample analyzer. The biological sample analyzer according to claim 1,
Each of the independent electrodes is connected to an I / O pad by a wiring,
Biological sample analyzer. The biological sample analyzer according to claim 3,
Further provided is a solution supply mechanism for introducing a solution into each of the plurality of recesses formed by the openings of the partition of the second layer and the openings of the first layer.
Biological sample analyzer. The biological sample analyzer according to claim 3,
Surrounding the outer periphery of the partition, further comprising a liquid tank support provided with an inflow port for flowing the conductive liquid and an outflow port for flowing out the inflowing conductive liquid,
Biological sample analyzer. The biological sample analyzer according to claim 8,
The liquid tank support includes a partition that divides the plurality of independent electrodes into two or more sections, and the inflow port and the outflow port corresponding to each of the sections are provided.
Biological sample analyzer. The biological sample analyzer according to claim 1,
The third layer includes a plurality of the openings, and includes a region exposed by the plurality of openings of the first layer on a surface opposite to a region where the membrane is exposed by the openings. ,
Biological sample analyzer. The biological sample analyzer according to claim 3,
The partition is higher than the plurality of independent electrodes,
Biological sample analyzer. A biological sample analysis method for simultaneously observing a plurality of changes in current when a sample passes through a nanopore,
The solution is formed in a droplet form so as not to contact each other with respect to the plurality of openings formed in the first layer laminated on the membrane on which the nanopores are formed and the plurality of recesses formed by the surface of the membrane. The steps of introducing
Introducing a solution into an opening provided in the substrate supporting the membrane,
By applying a voltage between a plurality of independent electrodes and a single common electrode provided across the membrane, forming nanopores in a plurality of partial regions of the membrane exposed by the opening,
A current value flowing through the nanopore when a biological sample contained in one of the solution introduced into the plurality of recesses and the solution introduced into the opening provided in the substrate passes through the nanopore. Measuring the
A biological sample analysis method comprising: The biological sample analysis method according to claim 12,
In the step of introducing a solution to the plurality of recesses, the solution to be introduced is different between one of the plurality of recesses and another one of the plurality of recesses,
Biological sample analysis method. The biological sample analysis method according to claim 12,
In the step of introducing a solution to the plurality of recesses, introducing the solution by a solution supply mechanism having a discharge port,
Biological sample analysis method. The biological sample analysis method according to claim 14,
In the step of introducing the solution to the plurality of recesses, the solution supply mechanism introduces an amount of the solution across at least two of the plurality of recesses,
further,
The solution supply mechanism includes a step of sucking the solution to prevent the solutions introduced into the plurality of recesses from coming into contact with each other,
Biological sample analysis method. The biological sample analysis method according to claim 12,
After the step of introducing the solution into the plurality of recesses, a voltage is applied between two adjacent independent electrodes of the plurality of independent electrodes, and a current value of a current flowing between the two adjacent independent electrodes is measured. Measuring
When the measured current value is equal to or more than a predetermined value, a step of determining a section to be measured by the adjacent two independent electrodes as a defective section,
A biological sample analysis method further comprising: The biological sample analysis method according to claim 12,
After the step of introducing the solution into the opening provided in the substrate, and as a step performed before the step of forming the nanopore,
Applying a voltage smaller than the voltage at the time of forming the nanopore between the independent electrode and the common electrode, measuring the current value of the current flowing between the independent electrode and the common electrode,
When the current value is equal to or more than a predetermined value, a step of determining the section that has measured the current value as a defective section,
A biological sample analysis method further comprising: The biological sample analysis method according to claim 12,
As a step performed after the step of forming the nanopore,
Applying a voltage, measuring a current value of a current flowing between the independent electrode and the common electrode,
When the current value is smaller than a predetermined value, a step of determining the section that has measured the current value as a defective section,
A biological sample analysis method further comprising: The biological sample analysis method according to claim 18, wherein
In the step of measuring the current value flowing through the nanopore, measuring the current value flowing through the nanopore when the biological sample passes through the nanopore in a section that is not the defective section,
Biological sample analysis method. A biological sample analysis method for simultaneously observing a plurality of changes in current when a sample passes through a nanopore,
A liquid tank support surrounding the plurality of openings with respect to a plurality of recesses formed by a plurality of openings provided in a first layer laminated on the membrane in which the nanopores are formed and a surface of the membrane; Introducing a solution from an inflow port provided in and filling with the solution;
Introducing an insoluble and insulating liquid with respect to the solution from the inflow port of the liquid tank support, allowing a part of the solution to flow out of an outflow port provided in the liquid tank support, So that the solution is in the form of a droplet that does not contact each other,
Introducing a solution into an opening provided in the substrate supporting the membrane,
By applying a voltage between a plurality of independent electrodes and a single common electrode provided across the membrane, forming nanopores in a plurality of partial regions of the membrane exposed by the opening,
A current value flowing through the nanopore when a biological sample contained in one of the solution introduced into the plurality of recesses and the solution introduced into the opening provided in the substrate passes through the nanopore. Measuring the
A biological sample analysis method comprising:

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