JP6955475B2 - Biological sample analyzer and biological sample analysis method - Google Patents

Description

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

Development of a blockage current type nanopore sequencer is underway as a biological sample analyzer for analyzing biological samples such as DNA and proteins. The blockage current type nanopore sequencer includes a thin film membrane having nanopores as large as a biological sample, and two liquid tanks having electrodes arranged above and below the membrane. The above two liquid tanks are filled with a solution, and a biological sample is introduced into one of the liquid tanks. Then, a voltage is applied to the electrodes, and the change in the current value flowing between the two electrodes is measured when the biological sample passes through the nanopore. The nanopore sequencer determines the structural characteristics of a biological sample by such measurement.

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

On the other hand, unlike biomolecules, the solid type uses a material with high mechanical strength for the membrane, so it can be a nanopore sequencer that is robust to environments 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 irradiation with an electron beam or application of a voltage. In a general solid nanopore sequencer, a material having high mechanical strength represented by a silicon nitride film is formed on a substrate, and then an opening is formed on the substrate to expose the silicon nitride film. As a result, a substrate having a membrane is formed. Then, the members constituting the liquid tank and the electrodes are attached above and below 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 the array of nanopore sequencers, it is necessary to parallelize not only the nanopores but also the peripheral structures such as the accompanying liquid tank and electrodes.

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

Japanese Unexamined Patent Publication No. 2012-26986

By the way, in the nanopore sequencer described in Patent Document 1, the electrodes independently provided in each sequencer are composed of a member different from the substrate having the membrane. Further, in the nanopore sequencer described in Patent Document 1, two flow paths for inflow and outflow are provided in order to supply a solution to each of a plurality of independently provided liquid tanks. With such a structure, the area of the nanopore sequencer increases depending on the flow path and the installation area of another member. As a result, it becomes a factor that hinders the integration and parallelization of sequencers.

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

In order to solve the above-mentioned problems, the membrane, a first layer in which a plurality of openings are provided so as to expose the membrane and laminated on one surface of the membrane, and the first layer of the first layer. The membrane has a plurality of independent electrodes provided in association with each of the plurality of openings, and is laminated on a second layer laminated on the first layer and on the other surface of the membrane. A third layer provided with a single opening so that the surface opposite to the region exposed by the plurality of openings of the first layer of the one surface is exposed, and the independent layer. The electrode is a common electrode provided on the opposite side of the membrane, and is formed by a recess formed by the opening of the first layer and the single opening of the third layer. A common electrode that causes a potential difference between the plurality of independent electrodes in a state where a conductive liquid is introduced into each of the recesses is provided, and the plurality of the first layer of the membrane is provided. Provided is a biological sample analyzer in which the portions exposed by the openings are insulated from each other.

Further, it is a biological sample analysis method for simultaneously observing a plurality of changes in current when a sample passes through a nanopore, and has a plurality of openings provided in a first layer laminated on a membrane on which the nanopore is formed. A step of introducing a solution from an inflow port provided in a liquid tank support surrounding the plurality of openings into a plurality of recesses formed by the surface of the membrane and filling the plurality of recesses with the solution, and a step of filling the liquid tank support with the solution. An insoluble and insulating liquid to the solution is introduced from the inlet, a part of the solution is discharged from the outlet provided in the liquid tank support, and the solution is discharged to the plurality of recesses. A step of forming a droplet-like shape that does not contact each other, a step of introducing a solution into an opening provided in a substrate supporting the membrane, and a plurality of independent electrodes provided across the membrane. By applying a voltage between one common electrode, a step of forming nanopores in a plurality of partial regions of the membrane exposed by the opening, and the solution introduced into the plurality of recesses and the substrate. Provided is a biological sample analysis method comprising a step of measuring a current value flowing through the nanopore when a biological sample contained in any one of the solution introduced into the opening provided passes through the nanopore. do.

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

FIG. 5 is a cross-sectional view of an analysis device included in the biological sample analyzer according to the first embodiment. It is a figure which shows schematic the whole structure of the biological sample analyzer which concerns on Example 1. FIG. It is a flowchart which shows the analysis flow of a biological sample using a biological sample analyzer. It is a figure which shows how the solution supply mechanism introduces various solutions into an analysis device. It is a figure which shows how the solution supply mechanism introduces various solutions into an analysis device. It is a figure which shows how the solution supply mechanism introduces various solutions into an analysis device. It is a figure which shows how the solution supply mechanism introduces various solutions into an analysis device. It is a figure which shows the state which introduced the biological sample into a common liquid tank. It is a top view of the analysis device when the number of arrays is 64 (8 rows, 8 columns). It is a figure for demonstrating the introduction method of the solution in Example 2. FIG. Example It is a top view of the analysis device when the number of arrays is 256 (arrays of (8 rows, 8 columns) are arranged in (2 rows, 2 columns)). It is sectional drawing of a part of the analysis device of Example 3. FIG. It is a figure which shows the state which the substrate opening 108A and the substrate opening 108B are separated by a support part. It is sectional drawing of the analysis device which concerns on Example 4. FIG. It is a figure which shows another example of a solution support.

Hereinafter, examples of the present disclosure will be described with reference to the drawings. Each drawing is drawn schematically, and unnecessary parts are omitted. The structures, materials and methods described in the examples are examples for embodying the ideas of the present disclosure, and do not strictly specify the materials and dimensions. Further, the examples of the present disclosure are not limited to the examples described later, and various modifications can be made within the scope of the technical idea.

<Example 1>
FIG. 1 is a cross-sectional view of an analysis device 10 included 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), wiring 103 (103A, 103B, 103C, 103D), and an independent device. The electrode 104 (104A, 104B, 104C, 104D), the I / O pad 105 (105A, 105B, 105C, 105D), the partition wall 106, the recess 107 (107A, 107B, 107C, 107D) and the common electrode 200 are provided. The substrate 100 is provided with a substrate opening 108, and the substrate opening 108 functions as a common liquid tank 201.

An insulating layer 102 (first layer) is laminated on one surface of the membrane 101, and a substrate 100 (third layer) is laminated on the other surface. The insulating layer 102 is provided with a plurality of openings 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, a plurality of independent electrodes 104 are provided above the membrane 101, and the membrane 101 is exposed at the bottom of the recess 107 formed by the insulating layer 102 and the partition wall 106. In addition, one independent electrode 104, one recess 107, and the partition wall 106 surrounding the recess 106 define one compartment. That is, the biological sample analyzer can simultaneously execute a plurality of biological sample analyzes as many as the number of compartments.

Further, each of the plurality of independent electrodes 104 is connected to the I / O pad 105 via a wiring 103 patterned in a state of being covered with the insulating layer 102. The partition wall 106 is one of the plurality of openings (for example, the first layer portion of the recess 107A) of the insulating layer 102 and one of the plurality of independent electrodes 104 associated with one of the plurality of openings (for example, the first layer portion of the recess 107A). For example, a pair of independent electrodes 104A) and 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. A pair of another 104 (eg, independent electrode 104B) is provided so as to isolate it from each other.

On the other hand, the substrate opening 108 and the common electrode 200 are arranged on opposite sides 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, and the common electrode. The material of the 200 and the I / O pad is gold, the material of the partition wall 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 and side surfaces of the partition wall 106 surrounding the membrane 101 are hydrophobic. Here, as an example, the diameter of the membrane 101 is 200 nm, the short dimension of the first electrode 104 is 1 μm, the opening diameter of the partition wall 106 is 25 μm, and the array pitch is 50 μm. The partition wall 106 does not have to be hydrophobic or sufficiently hydrophobic, for example, it may be one that has been subjected to surface hydrophobic treatment. In that case, the hydrophobic treatment may be applied only to the upper surface of the partition wall 106, or the hydrophobic treatment may be applied to the entire surface of the partition wall 106.

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

FIG. 2 is a diagram schematically showing the 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, a 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 also connected to the power supply and the control / detection data acquisition unit 400 via wiring. The control circuit unit 300 controls, for example, the voltage applied to the plurality of independent electrodes 104, and transfers the signal obtained during the 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. For example, a data analysis program and a device control program are recorded in the memory, and the CPU measures the measurement data by reading the program and controls various devices.

The solution supply mechanism 500 is arranged above the analytical device (structural portion shown in FIG. 1) and serves to supply the solution to the membrane 101. The solution supply mechanism 500 is controlled by, for example, a power supply and a control signal output by the control / detection data acquisition unit 400, and discharges a specified amount of liquid at a specified position. Further, the solution supply mechanism 500 can also function as a suction mechanism by operating the actuator or the motor in the reverse direction of the discharge.

[Analysis flow of biological sample]
FIG. 3 is a flowchart showing an analysis flow of a biological sample using the biological sample analyzer 1. In the following, the case where the number of arrays is four will be described, 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 recess 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 recess 107A, the solution supply mechanism 500 is first aligned with the recess 107A, and then the recess 107A is filled (the membrane 101A and the independent electrode). Introduce a solution 501 in an amount sufficient to immerse the 104A in the solution and not leak into the adjacent recess 107B. This step was also performed on the other recesses (107B, 107C and 107D), and as shown in FIG. 4B, all the recesses 107 were filled with the solution 501. Here, the solutions 501 introduced between the adjacent recesses 107 form parallel liquid pools (503A, 503B, 503C, 503D) that are blocked from contacting each other. The solution 501 contains an electrolyte such as potassium chloride. Further, the solution supply mechanism 500 may have a plurality of protrusions, and the solution may be introduced in parallel with each recess (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 the upper part of the parallel liquid pools (503A, 503B, 503C, 503D) from the solution supply mechanism 500, and the parallel liquid pools (503A, 503B, 503C) are introduced. , 503D). The presence of the insulating fluid 504 prevents trace amounts of liquid pools (503A, 503B, 503C, 503D) from drying out. Here, the insulating fluid 504 is, for example, an oily liquid and is insoluble in the solution 501.

(3) Step S13
Subsequently, as shown in FIG. 4D, the solution 501 is introduced into the common liquid tank 201. As a result, the top and bottom of the membrane 101 are filled with the solution. The order of the set of steps S11 and S12 and step S13 may be reversed. That is, the steps may be carried out 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 the leak current value is measured. For example, the leak current value between the independent electrode 104A and the independent electrode 104B, the leak current value between the independent electrode 104C and the independent electrode 104D is measured in parallel, and then the leak current value between the independent electrode 104B and the independent electrode 104C is measured. To measure. 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), nanopores are not formed on the membrane 101 in the nanopore forming step described later, or This is to identify the defective section where the leak has occurred because various defects such as signal noise are generated in the sample analysis process. Note that step S14 may be performed after step S11, and may be performed before step S13, for example.

(5) Step S15
In determining whether or not a leak current exists, a constant current value that does not interfere with the nanopore forming step and the sample analysis step described later is set as a threshold value, and the measured leak current value is compared with the threshold value. The threshold value is, for example, 10 pA, which is a value sufficiently smaller than the current flowing through the nanopore in step S21 described later when the applied voltage is 0.1 V. If the leak current value is equal to or greater than the threshold value in the determination in step S15, 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 provided in the power supply and the 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 the section in which the leak current is not detected is regarded as a non-defective section.

(6) Step S16
A lower voltage 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 leakage current between membranes. At this time, the applied voltage is, for example, a sweep voltage in the range of −1 to + 1V.

(7) Step S17
The intermembrane leak current is set at a constant level that does not interfere with the sample analysis step described later as a threshold value, and the measured intermembrane leak current value is compared with the threshold value. For example, if there is an initial defect such as the membrane being cracked in advance, 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 a value sufficiently smaller than the current flowing through the nanopore in step S21 described later. If the intermembrane leak current value is equal to or greater than the threshold value in the determination in 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 unit. If the intermembrane leak current value is smaller than the threshold value in the determination in step S17, the process proceeds to step S18, and the section in which the leak current is not detected is regarded 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 (101A, 101B, 101C, 101D) is subjected to 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 forming 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 the 200 and each of the independent electrodes (104A, 104B, 104C, 104D) is measured. The diameter of the formed nanopores increases as the current value flowing through the membrane 101 increases, and can be controlled by the current value (see WO2015 / 097765A). The treatment of step S19 is carried out because when the hydrophilicity of the membrane 101 is insufficient and the membrane 101 and the solution 501 do not come into sufficient contact with each other, the voltage is not normally applied above and below the membrane 101 and nanopores are not formed. Is.

(10) Step S20
It is determined whether or not the current value 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 the predetermined current value, that section is determined to be defective. After that, 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 parallel in a plurality of compartments (a plurality of exposed portions of the membrane 101). At this time, it is not necessary to perform the step of applying this voltage and the step of measuring the current in the section determined to be defective after the formation of the liquid tank described above. If the nanopore forming step and the current value measuring step are performed only on the non-defective product section, the nanopores can be formed efficiently. However, if this defect occurs due to air bubbles between the membrane 101 and the solution 501, it may be relieved by performing the work of removing the air bubbles. If there are bubbles between the membrane 101 and the solution 501, the analysis device 10 is placed in a reduced pressure environment to expand the volume of the bubbles, increase the buoyancy of the bubbles, and remove the bubbles. Return 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 biological sample 502 charged in the liquid occurs. As a result, the biological sample 502 is introduced into each nanopore and passes through the nanopore. The detected current value differs between before the biological sample passes through the nanopore and when the biological sample passes through the nanopore. This is because the nanopores are partially blocked and the resistance value of the nanopores changes depending on the cross-sectional area of the biological sample. From these measured current values, the structure of the biological sample is analyzed. The structural analysis of the sample by this blockage current measurement may be performed on a section that is not a defective section.

Through each of the above steps, a highly integrated structure in which parallel elements such as the membrane 101, the independent electrode 104, and the recess 107 are formed on the substrate 100 without a flow path is realized, and parallel nanopore formation and sample analysis are efficient. Realized.

The content described above is an example, and the biological sample analyzer 1 is not limited to the above configuration. As long as the alignment with the substrate 100 and the adjustment of the solution discharge amount do not pose a problem, it is not necessary to incorporate the solution supply mechanism 500 into the apparatus, and the solution may be discharged manually using a micropipette.

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

In addition, different types of solutions 501 and biological samples 502 can be introduced into each of the plurality of independent recesses 107. In that case, it is not always necessary to change the types 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. As a result, if the same sample 502 is mixed with the same solution 501 and a plurality of analyzes are performed, the analysis accuracy can be improved, and if the same sample 502 is analyzed under different solution conditions, multifaceted information can be obtained. By arranging different samples 502, the measurement throughput can be improved.

Further, when the same sample 502 is introduced into all the recesses 107 for analysis, the biological sample 502 may be introduced into the common liquid tank 201 side.
FIG. 5 is a diagram showing a state in which 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 by reversing the directions of the voltages applied to the independent electrode 104 and the common electrode 200, for example, as compared with the case where the biological sample 502 is introduced into the recess 107. ..

Further, 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 compartment, there is no partition wall 106. May be 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 in the same compartment and the independent electrode 104.

Further, when the number of wiring 103 connecting the independent electrode 104 and the I / O pad 105 is large and the wiring cannot be arranged in one layer, the layer in which the wiring 103 is provided may be multi-layered. Further, for example, the material of the membrane 101 is silicon nitride, but silicon oxide, graphene, graphite, an organic substance, a polymer material, or the like may be used. The material of the independent electrode 104 and the common electrode 200 was gold, but other metals such as silver and platinum may be used. The material of the partition wall 106 is a polyimide resin, but an insulating material such as an epoxy resin or silicon oxide may be used.

Further, although the substrate 100 is installed so that the membrane 101 is on the upper side, the substrate 100 may be located on the upper side of the membrane 101. In that case, a solution 501 having sufficient surface tension is selected so that a liquid tank is formed in the recess 107, and the solution 501 is sprayed from below toward the recess 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 depicts the layout of the substrate 100, the membrane 101, the I / O pad 105, the partition wall 106, and the substrate opening 108. The partition wall 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 (omitted in FIG. 6) shown in FIG. It is connected. Further, the substrate opening 108 formed on the back surface side of the substrate 100 has a surface opposite to the region exposed by the plurality of openings of the insulating layer 102 (first layer) on one surface of the membrane 101. It is provided to be exposed.

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

Further, 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 recesses (for example, between the recess 107A and the recess 107B). It is important that they do not touch each other). In order to improve 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 mixed solution of hydrogen sulfide and hydrogen peroxide to remove organic substances accumulated on the surface of the membrane 101, and alcohol may be added before the solution 501 is introduced. It may be introduced and replaced with solution 501, or the analytical device 10 may be treated with oxygen plasma. Further, the above methods may be combined.

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

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

FIG. 7 is a diagram for explaining a method of introducing the solution 501 in the case of Example 2. As shown in FIG. 7, in the second embodiment, the introduction of the solution 501 into each recess 107 in step S11 is simultaneously executed in the recess 107A and the recess 107B by using the solution supply mechanism 500.

In this case, first, the shape of the solution 501 is a shape that straddles the recess 107A and the recess 107B. Then, 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 wall 106 located between the membranes 101A and 101B is a hydrophobic surface, so that when the solution 501 is aspirated, the solution 501 becomes two. It splits and remains in the recesses 107A and 107B in the form of a pool so that the solutions 501 do not come into contact with each other. When sucking the solution 501, for example, a motor or the like provided in the solution supply mechanism 500 is operated in the reverse direction of the discharge.

By the discharge operation and the suction operation, the exposed portion of the membrane 101A and the independent electrode 104A in the recess 107A and the exposed portion and the independent electrode 104B of the membrane 101B in the recess 107B are filled with the solution 501. The solution 501 forms a parallel liquid pool independent of each other between the adjacent recesses 107A and 107B. The above process is also performed on the recess 107C and the recess 107D to form a liquid pool in the entire array. When the method for introducing the solution 501 of Example 2 is applied, the same sample and the same solution can be measured at the same time in the compartments (for example, the recess 107A and the recess 107B) in which the solution 501 is introduced all at once, so that the measurement accuracy is high. Not only is it obtained, but the solution 501 is introduced into a plurality of compartments at the same time, so that the introduction throughput of the solution 501 is high.

<Example 3>
Hereinafter, a device structure in which the membrane 101 is less likely to be damaged and the defect rate is reduced as compared with the analytical device 10 according to the first embodiment will be described. As shown in FIG. 4, the analysis device 10 according to the first embodiment has four recesses (107A, 107B, 107C, 107D) and one substrate opening 108, and four recesses (107A). , 107B, 107C, 107D) are supported only by the substrate 100 on the outer periphery of the array. Further, 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 wall 106. Therefore, when the number of arrays increases and the area of the substrate opening 108 increases, the portion exposed by the substrate opening 108 of the membrane 101 becomes stressed by the insulating layer 102 and the partition wall 106 laminated on the substrate opening 108 and external foreign matter. It is easily damaged by contact with.

The biological sample analyzer according to the third embodiment has a different structure of the analytical device from the biological sample analyzer 1 according to the first embodiment. Other than that, the structure and method described in Example 1 are applied, and the description of the same steps and structures as in Example 1 is omitted.

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

FIG. 9 is a cross-sectional view of a part of the analysis device 20 of the third embodiment. In FIG. 9, the exposed portions 101A and 101B of the membrane are encapsulated in the same substrate opening 108A, and the exposed portions 101C and 101D of the membrane are encapsulated in the same substrate opening 108B. As shown in FIG. 9, there is a convex portion between the substrate opening 108A and the substrate opening 108B, and the strength of the substrate 100 is maintained. Further, 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 showing a state in which the substrate opening 108A and the substrate opening 108B are partitioned 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, and the device area is not increased. .. 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 portion S, respectively. By doing so, the measurement throughput can be improved.

<Example 4>
In the biological sample analyzer 1 of Example 1, the solution 501 and the biological sample 502 were introduced into a plurality of recesses 107 by using the solution supply mechanism 500. In the biological sample analyzer according to Example 4, the solution 501 and the biological sample 502 are introduced into the analytical device by a method different from that of Example 1. Since the fourth embodiment is the same as the first embodiment except for the method of introducing the solution 501, the description of the same steps and structures will be omitted. Specifically, in the flowchart shown in FIG. 3, only the steps of the first steps S11 and S12 are different between the first and fourth embodiments. Therefore, only the steps of 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 analytical 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 Example 4, in step S11 in FIG. 3, the solution 501 and the biological sample 502 are introduced into the solution support 505 to introduce the solution 501 and the biological sample 502 into each of the plurality of recesses 107.

FIG. 11A is a diagram showing how the liquid tank support 505 is filled with the solution 501. In the case of the biological sample analyzer of Example 4, 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 held at the bottom. The solution is supplied to the recesses (107A, 107B, 107C, 107D). In this step S11, the exposed portions (101A, 101B, 101C, 101D) of the membrane are also filled with the solution 501.

FIG. 11B is a diagram showing a state in which the insulating fluid is introduced into the liquid tank support 505. In the case of the biological sample analyzer of Example 4, in step S12, the insulating liquid 504 is poured from the opening of the liquid tank support 505. At least the independent electrodes (104A, 104B, 104C, 104D) and the exposed parts of the membrane (101A, 101B, 101C, 101D) are sufficiently hydrophilic and between each of the plurality of recesses (107A, 107B, 107C, 107D). Since the partition wall 106 arranged in is a hydrophobic surface, the solution 501 is torn by the inflow of the insulating liquid 504, and a liquid pool (503A, 503A, which does not contact each other in a plurality of recesses (107A, 107B, 107C, 107D)). 503B, 503C, 503D) can be done. In other words, each of the exposed portions 101 of the membrane contained in each of the plurality of recesses 107 and each of the plurality of independent electrodes 104 are filled with the solution 501, and between adjacent recesses (for example, the recess 107A and the recess). The solution is blocked (between 107B). By using the above method, the solution 501 can be introduced into a plurality of recesses 107 at the same time, so that the introduction throughput of the solution 501 is high. When the insulating fluid is introduced, the liquid 501 is prevented from being torn off due to the insulating fluid overflowing from the discharge port through the upper part in the liquid tank support 505 and being introduced into the recess 107. The tank support 505 needs to be designed at a sufficiently low height.

FIG. 12 is a diagram showing another example of solution support. As shown in FIG. 12, the liquid tank support 505 may be provided with a partition that separates the compartments of the array (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 analytical device is not increased. The method of partitioning the liquid tank support 505 is not limited to this. In the liquid tank support 505 having the partition, different solutions and / or different biological samples may be introduced into each of the plurality of partitioned liquid tank supports 505. The analytical devices shown in FIGS. 11 and 12 do not require the solution supply mechanism 500, and the cost of the device can be reduced.

As described above, the biological sample analyzer of the present disclosure eliminates the flow path in the array of the solid nanopore sequencer, and integrates the liquid tank, electrodes (independent electrode and common electrode), etc. in a single analytical device. Can be parallelized and parallelized. Therefore, the biological sample analyzer of the present disclosure can not only make the device compact, but also improve the measurement throughput, for example.

The present disclosure is not limited to the above-described embodiment, and includes various modifications. For example, the above-described embodiment has been described in detail in order to explain the present disclosure in an easy-to-understand manner, and is not necessarily limited to the one including all the configurations described. Further, it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. Further, it is possible to add / delete / replace a part of the configuration of each embodiment with another configuration.

100: Substrate 101 (101A, 101B, 101C, 101D): Membrane 102: Insulation layer 103 (103A, 103B, 103C, 103D): Wiring 104 (104A, 104B, 104C, 104D): Independent electrode 105 (105A, 105B, 105C, 105D): I / O pad 106: partition wall 107 (107A, 107B, 107C, 107D): recess 108, 108A, 108B: substrate opening 200: second electrode 201, 201A, 201B: common solution 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 (15)

Membrane and
A first layer, in which a plurality of openings are provided so that the membrane is exposed and laminated on one surface of the membrane,
A second layer having a plurality of independent electrodes provided in association with each of the plurality of openings of the first layer and laminated on the first layer.
A single opening laminated on the other surface of the membrane so that the opposite surface of the region exposed by the plurality of openings of the first layer of the one surface of the membrane is exposed. The third layer with the parts and
The independent electrode is a common electrode provided on the opposite side of the membrane, and has a plurality of recesses formed by the openings of the first layer and the single electrode of the third layer. A common electrode that causes a potential difference between the plurality of independent electrodes in a state where a conductive liquid is introduced into each of the recesses formed at the openings, and a common electrode.
A solution supply mechanism for introducing the conductive liquid into the plurality of recesses formed by the openings of the first layer, and
With
Among the membrane, portions exposed by the plurality of openings of the first layer are insulated from each other,
The solution supply mechanism introduces the conductive liquid in an amount that straddles at least two of the plurality of recesses, sucks the conductive liquid, and introduces the conductive liquid into the plurality of recesses. Do not touch 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 hydrophobic surrounding one pair of the plurality of independent electrodes associated with one of the plurality of openings and one of the plurality of openings. Has a sex area,
Biological sample analyzer. The biological sample analyzer according to claim 1.
The second layer includes one pair 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. Further having an insulating partition that separates another one of the portions and another pair of the plurality of independent electrodes associated with the other one of the plurality of openings from each other.
Biological sample analyzer. In the biological sample analyzer according to claim 3,
The upper surface of the partition wall is hydrophobic,
Biological sample analyzer. In the biological sample analyzer according to claim 4,
The sides of the partition are hydrophobic,
Biological sample analyzer. In the biological sample analyzer according to claim 1,
Each of the independent electrodes is connected to the I / O pad by wiring.
Biological sample analyzer. In the biological sample analyzer according to claim 3,
Wherein the plurality of recesses are formed in the opening where the barrier ribs and the first layer and the second layer has has,
Biological sample analyzer. In the biological sample analyzer according to claim 1,
The third layer includes a plurality of the openings, and the surface opposite to the region where the membrane is exposed by the openings includes a region exposed by the plurality of openings of the first layer. ,
Biological sample analyzer. In the biological sample analyzer according to claim 3,
The partition wall is taller than the plurality of independent electrodes.
Biological sample analyzer. This is a biological sample analysis method that simultaneously observes multiple changes in current as a sample passes through a nanopore.
A solution in the form of droplets so as not to contact each other with respect to the plurality of openings provided 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. And the steps to introduce
The step of introducing the solution into the opening provided in the substrate supporting the membrane, and
A step of 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 provided across the membrane and a single common electrode.
The value of the current flowing through the nanopore when the biological sample contained in either 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. Steps to measure and
Only including,
In the step of introducing the solution into the plurality of recesses, the solution is introduced in an amount straddling at least two of the plurality of recesses by a solution supply mechanism having a discharge port, and then the solution is sucked to the plurality of recesses. Prevent the solutions introduced into the recesses from coming into contact with each other.
Biological sample analysis method. The biological sample analysis method according to claim 10.
In the step of introducing a solution into the plurality of recesses, the solution to be introduced differs between one of the plurality of recesses and another of the plurality of recesses.
Biological sample analysis method. The biological sample analysis method according to claim 10.
After the step of introducing the solution into the plurality of recesses, a voltage is applied between two adjacent independent electrodes among the plurality of independent electrodes, and the current value of the current flowing between the two adjacent independent electrodes. And the steps to measure
A step of determining a section to be measured by the two adjacent independent electrodes when the measured current value is equal to or higher than a predetermined value, and a step of determining a defective section.
A biological sample analysis method further comprising. The biological sample analysis method according to claim 10.
As a step performed after the step of introducing the solution into the opening provided in the substrate and before the step of forming the nanopores.
A step of applying a voltage smaller than the voltage at which the nanopore is formed between the independent electrode and the common electrode and measuring the current value of the current flowing between the independent electrode and the common electrode.
A step of determining a section in which the current value is measured as a defective section when the current value is equal to or higher than a predetermined value, and
A biological sample analysis method further comprising. The biological sample analysis method according to claim 10.
As a step performed after the step of forming the nanopore,
A step of applying a voltage and measuring the current value of the current flowing between the independent electrode and the common electrode, and
A step of determining a section in which the current value is measured as a defective section when the current value is smaller than a predetermined value, and
A biological sample analysis method further comprising. The biological sample analysis method according to claim 14.
In the step of measuring the current value flowing through the nanopore, the current value flowing through the nanopore when the biological sample passes through the nanopore in a section other than the defective section is measured.
Biological sample analysis method.

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