Biological sample analyzer

Abstract

The purpose of the present invention is to provide a technique for biological sample analysis using pores, wherein a biological sample is measured at an appropriate passage frequency regardless of whether the sample density is high or low. The biological sample analysis device according to the present disclosure comprises first and second chambers disposed facing each other via a substrate having pores, the first chamber being partitioned into a first section and a second section, and the liquid replacement efficiency when a liquid in the first section is replaced with other liquid being lower than the liquid replacement efficiency when the liquid in the second section is replaced with other liquid (see FIG. 1)

Description

[Technical field]

[0001] The present disclosure relates to techniques for analyzing biological samples. [Background technology]

[0002] In the field of next-generation DNA sequencers, a method that electrically measures the base sequence of a biomolecule (hereinafter referred to as "DNA": deoxyribonucleic acid) directly without performing an extension reaction or fluorescent labeling is attracting attention. Specifically, research and development of a nanopore DNA sequencing method is being actively carried out. This method determines the base sequence by directly measuring the DNA chain without using reagents.

[0003] In this nanopore DNA sequencing method, the base sequence is measured by measuring the blocking current that occurs when a DNA strand passes through a pore (hereafter referred to as "nanopore") formed in a thin film. In other words, the blocking current changes depending on the difference in the individual base types contained in the DNA strand, so the base types can be identified sequentially by measuring the amount of blocking current. In this method, no enzymatic amplification of the template DNA is performed, and no labels such as fluorescent substances are used. Therefore, it has high throughput, low running costs, and enables DNA decoding of long bases.

[0004] A biomolecule analysis device used in analyzing DNA in a nanopore DNA sequencing method generally includes first and second liquid tanks filled with an electrolyte solution, a thin film separating the first and second liquid tanks, and first and second electrodes provided in the first and second liquid tanks. The biomolecule analysis device can also be configured as an array device. An array device is a device that includes multiple sets of liquid chambers separated by thin films. For example, the first liquid tank is a common tank, and the second liquid tank is multiple individual tanks. In this case, electrodes are provided in each of the common tank and the individual tanks.

[0005] In this configuration, a voltage is applied between the first and second liquid tanks, and an ionic current flows through the nanopore according to the nanopore diameter. A potential gradient is also formed in the nanopore according to the applied voltage. When a biomolecule is introduced into the first liquid tank, the biomolecule is sent to the second liquid tank through the nanopore according to the diffusion of the biomolecule and the generated potential gradient. At this time, analysis of the biomolecule is performed according to the blocking rate when each nucleic acid blocks the nanopore. The biomolecule analyzer has a measurement unit that measures the ionic current (blocking signal) flowing between electrodes provided in the biomolecule analysis device, and obtains sequence information of the biomolecule based on the value of the measured ionic current (blocking signal).

[0006] One of the challenges of the nanopore DNA sequencing method is that the range of DNA concentrations to be measured is limited, so the concentration must be adjusted beforehand. If the concentration of DNA input is too high, it will clog the nanopore and measurement will be impossible. Conversely, if the concentration is too low, the frequency with which DNA passes through the nanopore will decrease, and it will take time to obtain the required amount of data. Therefore, in general, the DNA concentration is adjusted to be within the appropriate range before performing nanopore sequencing. This has the disadvantage of increasing the amount of work required by the user.

[0007] Patent Document 1 uses an electrode with a probe that specifically binds to the target substance. In this patented configuration, even if the sample is at a low concentration, the sample binds to the probe, increasing the concentration near the nanopore and improving sensitivity.

[0008] Patent Document 2 uses a method of providing an electrode near a nanopore. In this patent configuration, even if the sample is at a low concentration, the electric field generated near the nanopore causes the sample to aggregate, thereby increasing the frequency of passing through.

[0009] Patent Document 3 uses a method of guiding a biological sample to a nanopore by Benard convection. In this patent configuration, Benard convection is generated by the temperature difference of the nanopore device, and the biological molecules are stirred in the solution and guided to the nanopore, thereby increasing the frequency of passing the biological molecules.

[0010] Patent Document 4 aims to suppress the passage of a modifier molecule alone through a nanopore by adjusting the pH of the electrolyte solution, thereby suppressing background noise caused by the modifier molecule alone. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] JP 2019-045261 A [Patent Document 2] JP 2013-036865 A [Patent Document 3] Patent No. 6498314 [Patent Document 4] JP 2020-085578 A Summary of the Invention [Problem to be solved by the invention]

[0012] When using the technology of Patent Document 1, when a voltage is applied for detection, not only the probe bound to the sample to be measured but also the probe not bound to the sample passes through the nanopore. This causes background noise from the probe itself to be included in the data to be measured. In addition, although it is possible to increase the concentration of the sample to be measured near the nanopore, it is not possible to adjust it to dilute the sample when there is a large amount of sample.

[0013] When using the technology of Patent Document 2, electrodes are required to increase the potential near the nanopore, which complicates the device configuration. In the case of multi-channels, the measurement control of the system becomes complicated. Also, like Patent Document 1, it is possible to increase the concentration of the sample to be measured near the nanopore, but conversely, it is not possible to adjust it to dilute the sample when there is a large amount of sample.

[0014] When using the technology of Patent Document 3, a mechanism such as a temperature control system that forms a temperature gradient to generate Benard convection is necessary, making the device configuration complicated. Also, like Patent Document 1, it is possible to increase the concentration of the sample to be measured near the nanopore, but conversely, it is not possible to adjust the concentration to dilute the sample when there is a large amount of sample.

[0015] Patent Document 4 aims to reduce background noise caused by single modified molecules, but does not give any special consideration to a method for measuring samples at an appropriate passing frequency regardless of whether the biological sample concentration is high or low.

[0016] The present disclosure has been made in consideration of the above-mentioned problems, and aims to provide a technology for biological sample analysis using nanopores that can measure samples at an appropriate passing frequency regardless of whether the biological sample concentration is high or low. [Means for solving the problem]

[0017] The biological sample analysis device according to the present disclosure comprises first and second chambers arranged opposite each other via a substrate having fine holes, the first chamber being divided into a first compartment and a second compartment, and the liquid replacement efficiency when replacing the liquid in the first compartment with another liquid is lower than the liquid replacement efficiency when replacing the liquid in the second compartment with another liquid. Effect of the Invention

[0018] The biological sample analyzer according to the present disclosure can perform measurements at an appropriate pass frequency in a biological sample analyzer using pores, and can measure not only low-concentration samples but also high-concentration samples, thereby expanding the dynamic range. [Brief description of the drawings]

[0019] [Figure 1] 1 is a diagram showing an example of the configuration of a biological sample analyzer 100 according to a first embodiment. [Diagram 2] The configuration in the vicinity of the nanopore when the concentration of the biological sample 113 is high is shown. [Diagram 3] The configuration in the vicinity of the nanopore when the concentration of the biological sample 113 is low is shown. [Figure 4A] FIG. 2 is a diagram showing the flow channel structure of a flow cell. [Figure 4B] FIG. 4B is an enlarged view of the nanopore substrate 103 portion of FIG. 4A. [Figure 5A] The compartments for evaluating the liquid exchange efficiency are shown. [Figure 5B] The compartments for evaluating the liquid exchange efficiency are shown. [Figure 6A] The time course of the liquid replacement rate of the section a 520 and the section b 521 is shown. [Figure 6B] The time course of the liquid replacement rate of the section a 520 and the section b 521 is shown. [Figure 7] FIG. 13 is a diagram showing a schematic diagram of the change over time in the liquid replacement rate of section a520. [Figure 8] FIG. 13 is a diagram showing a schematic diagram of the change over time in the liquid replacement rate of section a520. [Figure 9A] 13 shows a configuration example near a nanopore in the second embodiment. [Figure 9B] 13 shows another example of the configuration near the nanopore in the second embodiment. [Figure 10A] 13 shows a configuration example near a nanopore in embodiment 3. [Figure 10B] 13 shows another example of the configuration near the nanopore in embodiment 3. [Figure 11A] FIG. 1 is a schematic diagram of an experimental system combining Embodiments 1 to 3. [Figure 11B] 11B is an enlarged view showing the structure of the nanopore substrate 103 and the compartment forming portion 117 in FIG. 11A. [Figure 11C] 11B is an enlarged view showing the structure of the nanopore substrate 103 and the compartment forming portion 117 in FIG. 11A. [Figure 12] The experimental results using the experimental system of FIG. 11A are shown. [Figure 13A] FIG. 11 is a top view of a flow cell according to a fourth embodiment. [Figure 13B] 1 is a three-dimensional view of a flow channel 104. FIG. [Figure 13C] FIG. 2 is an enlarged view of the periphery of the nanopore 102. [Figure 14A] The time course of the liquid replacement rate in section c1323 is shown. [Figure 14B] The time course of the liquid replacement rate in section c1323 is shown. [Figure 15] FIG. 13 is a schematic diagram showing the change over time in the liquid replacement rate of section c1323 when the flow rate is 3 μL / s. [Figure 16] 13 shows another example of a configuration for forming a concentration gradient between channels. [Figure 17A] 13 shows another example of a configuration for forming a concentration gradient between channels. [Figure 17B] 13 shows another example of a configuration for forming a concentration gradient between channels. [Figure 18] 13 shows a configuration example in the vicinity of a nanopore in embodiment 5. [Figure 19] 13 shows an example of the configuration in the vicinity of a nanopore of a biological sample analyzer 100 according to a sixth embodiment. [Figure 20] 13 shows the results of a simulation of the time course of the liquid replacement efficiency of the section c1323 in the seventh embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Hereinafter, a biological sample analysis technique according to an embodiment of the present disclosure will be described in detail with reference to the drawings. The biological sample analysis technique according to the present embodiment relates to a technique for analyzing nucleic acids, such as DNA and RNA (ribonucleic acid), and to a technique for efficiently passing a biopolymer through a nanopore.

[0021] More specifically, the present invention relates to a technology for allowing nucleic acids to pass through a nanopore at an appropriate frequency, for example for nucleic acid sequencing using a nanopore sequencer, and more specifically, in a multi-channel nanopore sequencer, the dynamic range is expanded by making the nucleic acid concentration non-uniform through differences in the liquid replacement efficiency between each compartment of the nanopore.

[0022] <Embodiment 1: Explanation of nucleic acid molecule measurement> 1 is a diagram showing a configuration example of a biological sample analyzer 100 according to embodiment 1 of the present disclosure, and exemplarily shows a cross-sectional configuration of a nanopore substrate and an observation container in which the nanopore substrate is disposed. As shown in Fig. 1, an observation container (chamber section) 101 for biological sample analysis has two closed spaces, namely, a sample introduction section 104 and a sample outflow section 105, separated by a nanopore substrate (substrate) 103 having a nanopore 102.

[0023] The sample introduction section 104 and the sample outflow section 105 are divided into pores by a section forming section 117. The sample outflow section 105 may be divided by providing a separate partition member instead of or in combination with the section forming section 117. In FIG. 1, four nanopores 102A, 102B, 102C, and 102D are provided, but the number is not limited to two or more. The sample introduction section 104 and the sample outflow section 105 are disposed adjacent to each other and facing each other with the substrate 103 interposed therebetween. The sample introduction section 104 and the sample outflow section 105 are in communication with each other through the nanopore 102.

[0024] The sample introduction section 104 and the sample outflow section 105 are filled with liquids 110 and 111 introduced through inflow channels 106 and 107 connected to the two sections, respectively. The liquids 110 and 111 flow out from outflow channels 108 and 109 connected to the sample introduction section 104 and the sample outflow section 105. In this manner, the sample introduction section 104 and the sample outflow section 105 also function as flow channels. The inflow channels 106 and 107 may be provided at positions adjacent to each other (opposing each other) with the nanopore substrate 103 in between, but are not limited to this arrangement. The outflow channels 108 and 109 may be provided at positions adjacent to each other (opposing each other) with the nanopore substrate 103 in between, but are not limited to this arrangement. The set of the inflow channels 106 and 107 and the outflow channels 108 and 109 may be one or more, and may be the same number as the number of pores or more.

[0025] The liquid (solvent) 110 is preferably a sample solution containing a biological sample 113 to be analyzed. The liquid 110 preferably contains a large amount of ions that carry electric charge (hereinafter referred to as "ionic liquid"). The liquid 110 preferably contains only an ionic liquid in addition to the biological sample. As the ionic liquid, an aqueous solution in which an electrolyte with a high degree of ionization is dissolved is preferable, and a salt solution, for example, an aqueous solution of potassium chloride, can be suitably used. The melting point of the liquid (solvent) 110 may be less than 0 degrees. The biological sample 113 is preferably one that has an electric charge in the ionic liquid. The biological sample 113 is typically a nucleic acid molecule, but is not limited thereto, and may be a biological sample such as a peptide, a protein, a cell, a blood cell, or a virus. The biological sample shown here is not limited thereto.

[0026] The sample introduction section 104 and the sample outflow section 105 are provided with, for example, electrodes 114 and 115 arranged to face each other with the nanopore 102 in between. In this embodiment, a voltage application section 116 is provided to apply a voltage to the electrodes 114 and 115. By applying a voltage to the electrodes 114 and 115, the charged biological sample 113 passes from the sample introduction section 104 through the nanopore 102 and moves to the sample outflow section 105. The electrodes 114 and 115 and the voltage application section 116 constitute a biological sample induction section that causes the charged biological sample 113 to pass from the sample introduction section 104 through the nanopore 102 and move to the sample outflow section 105. These constitute a blockage current detection section (detection section). Hereinafter, they are also referred to as blockage current detection sections (detection sections) 114 and 115.

[0027] When a nucleic acid molecule passes through a nanopore, it blocks the flow of ions in the nanopore 102, causing a decrease in current (blockage current). By measuring the magnitude of this blocking current and the duration of the blocking current using known blocking current detection units (detection units) 114 and 115, it is possible to detect the length of each nucleic acid molecule passing through the nanopore 102. By providing blocking current detection units (detection units) 115A, B, C, and D for the number of nanopores, it is possible to measure the current value for each pore. The sample outflow compartments 105 are insulated from each other for each nanopore 102 by the compartment formation unit 117, and the current flowing through each nanopore 102 can be measured independently. It is also possible to distinguish the type of each base constituting a nucleic acid molecule.

[0028] In Figure 1, the upper part of the chamber section 101 is the sample introduction section 104 and the lower part is the sample outflow section, but the lower part may be the sample introduction section 104 and the upper part may be the sample outflow section 105, and the biological sample 113 passing through the nanopore 102 may be detected.

[0029] Before the measurement of the biological sample, a solution (blank solution) different from the biological sample solution to be measured is used as the liquid 110 for the purposes of pore preservation, pore pretreatment, pore quality judgment, blank measurement, etc. Therefore, at the time of measurement, this solution is replaced with the target biological sample solution.

[0030] In this embodiment, the distance between the partition forming parts 117 (i.e., the opening size of each partition separated by the partition forming parts 117) is not uniform, and the volume of the liquid 110 surrounded by the nearby partition forming parts 117, and therefore the liquid inflow efficiency, differs in the vicinity of each of the nanopores 102A to D. As a result, for example, when the liquid 110 is replaced from a blank solution to a solution containing a biological sample 113, the liquid replacement efficiency in the vicinity of each of the nanopores 102A to D differs from partition to partition, and therefore the biological sample concentration in the vicinity of the nanopore differs from partition to partition.

[0031] FIG. 2 shows the configuration near the nanopore when the concentration of the biological sample 113 is high. The volume of the liquid 110 near the nanopore 102B surrounded by the partition forming portion 117 is larger than that near the nanopore 102A surrounded by the partition forming portion 117. In other words, the opening size of the nanopore 102A partition (first partition) is smaller than the opening size of the nanopore 102B partition (second partition). This results in a high liquid replacement efficiency near the nanopore 102B, and a high concentration of the biological sample 113 near the nanopore. Therefore, if the original biological sample concentration is too high, clogging occurs, making it impossible to perform sample measurement. On the other hand, the concentration of the biological sample 113 is relatively low near the nanopore 102A, where the liquid replacement efficiency is low, and clogging can be avoided and measurement can be performed.

[0032] The vicinity of the nanopore here refers to a region located closer to the nanopore 102 side than the opening of the compartment. In other words, the liquid exchange efficiency in a region (first region) closer to the nanopore 102A than the opening of the nanopore 102A compartment (first compartment) is lower than the liquid exchange efficiency in a region (second region) closer to the nanopore 102B than the opening of the nanopore 102B compartment (second compartment). It is not necessary that such a difference in liquid exchange efficiency occurs for all regions within the compartment, and it is sufficient that at least a clear difference in blockage current occurs between nanopores 102A and 102B.

[0033] 3 shows the configuration near the nanopore when the concentration of the biological sample 113 is low. Because the liquid replacement efficiency is low near the nanopore 102A, the concentration of the biological sample 113 near the nanopore becomes too low, the frequency at which the sample passes through the nanopore 102A decreases, and it takes a long time to acquire a specified amount of data. On the other hand, near the nanopore 102B, where the liquid replacement efficiency is high, the concentration of the biological sample 113 does not become low, and measurement can be performed with an appropriate passing frequency.

[0034] In a nanopore array device, if the structure near all pores is uniform, then a high-concentration sample will cause clogging in all pores, and a low-concentration sample will cause a decrease in the frequency of passing through, lengthening the measurement time required to obtain the required data. To avoid this, it is necessary to take the time to pre-adjust the solution concentration to the recommended concentration before performing the measurement. In contrast, if the structure is such that the concentration near the nanopores differs for each pore, as in this embodiment, there will be a certain number of pores that can perform measurements at an appropriate frequency, regardless of whether the sample concentration is high or low.

[0035] The number of nanopores is not limited to the structure of this embodiment as long as it is 2 or more. For example, two types of structures with different liquid replacement efficiencies may be alternately arranged in the array device, or the liquid replacement efficiency may vary by the number of pores.

[0036] <Embodiment 1: Description of the container> The container used in this embodiment has a chamber section 101 and a nanopore substrate 103 disposed therein. The nanopore substrate 103 has a base material, a thin film formed facing the base material, and a nanopore 102 (which communicates with a sample introduction section 104 and a sample outflow section 105) disposed in the thin film. The nanopore substrate 103 is disposed between the sample introduction section 104 and the sample outflow section 105 of the chamber section 101. The nanopore substrate 103 may have an insulating layer. The nanopore substrate 103 is a thin film such as a solid substrate or a lipid bilayer. The outer periphery of the inner bottom surface of the second chamber, which is the sample outflow section 105, may be rounded. The nanopore substrate 103 may be formed from an electrically insulating material, for example, an inorganic material and an organic material (including a polymer material) or a lipid bilayer composed of an amphiphilic molecule layer. Examples of the electrical insulator material constituting the nanopore substrate 103 and the compartment forming portion 117 include silicon, silicon compounds, glass, quartz, polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), polystyrene, polypropylene, etc. Silicon compounds include silicon nitride, silicon oxide, silicon carbide, etc. In particular, the base (substrate) constituting the support portion of the substrate can be made of any of these materials, and may be, for example, silicon or a silicon compound.

[0037] The size and thickness of the nanopore substrate 103 and the compartment forming portion 117 are not particularly limited as long as they can provide the nanopore 102. The nanopore substrate 103 and the compartment forming portion 117 can be fabricated by methods known in the art or can be obtained commercially. For example, they can be fabricated using techniques such as photolithography or electron beam lithography, etching, laser ablation, injection molding, casting, molecular beam epitaxy, chemical vapor deposition (CVD), dielectric breakdown, electron beam or focused ion beam. The nanopore substrate 103 and the compartment forming portion 117 may be coated to prevent adsorption of non-target molecules to the surface.

[0038] The nanopore substrate 103 has at least one nanopore 102. The nanopore 102 is specifically provided in a thin film, but may be provided in a base (substrate) and an insulator at the same time in some cases. In this embodiment, the terms "pore", "nanopore" and "pore" refer to, for example, a hole of nanometer (nm) size (i.e., diameter of 1 nm or more and less than 1 μm) or micrometer (μm) size (i.e., diameter of 11 μm or more), which penetrates the nanopore substrate 103 and communicates between a sample introduction section and a sample outflow section. Alternatively, the nanopore substrate 103 may have a structure in which a protein having a pore in the center is embedded in the lipid bilayer nanopore substrate 103 (bio-type nanopore). The sizes of the holes shown here are not limited to these.

[0039] The nanopore substrate 103 preferably has a thin film for providing the nanopore 102. That is, the nanopore 102 can be simply and efficiently provided in the substrate 103 by forming a thin film of a material and thickness suitable for forming nano-sized holes on the substrate. In terms of nanopore formation, the material of the thin film is preferably, for example, graphene, silicon oxide (SiO2), silicon nitride (SiN), silicon oxynitride (SiON), metal oxide, metal silicate, etc. The thin film (and the entire substrate in some cases) may be substantially transparent. Here, "substantially transparent" means that the external light can be transmitted by approximately 50% or more, preferably 80% or more. The thin film may be a single layer or multiple layers. The thickness of the thin film is 0.1 nm to 200 nm, preferably 0.1 nm to 50 nm, and more preferably 0.1 nm to 20 nm. The thin film can be formed on the substrate by a technique known in the art, for example, low pressure chemical vapor deposition (LPCVD).

[0040] An insulating layer may be provided on the thin film. The thickness of the insulating layer is preferably 5 nm to 50 nm. Any insulating material may be used for the insulating layer, but it is preferable to use, for example, silicon or a silicon compound (silicon nitride, silicon oxide, etc.). In this embodiment, the "opening" of the nanopore or pore refers to the opening circle of the nanopore or pore at the portion where the nanopore or pore contacts the sample solution. When analyzing biopolymers, biopolymers, ions, etc. in the sample solution enter the nanopore 102 from one opening and exit the nanopore 102 from the same or opposite opening.

[0041] <First embodiment: Explanation of nanopore> The size of the nanopore 102 can be selected appropriately depending on the type of biopolymer to be analyzed. The nanopore 102 may have a uniform diameter, or may have different diameters depending on the site. The nanopore 102 may be connected to a pore having a diameter of 1 μm or more.

[0042] The nanopore 102 provided in the thin film of the nanopore substrate 103 has a minimum diameter portion, i.e., the smallest diameter of the nanopore 102, of 100 nm or less, for example, 1 nm to 100 nm, preferably 1 nm to 50 nm, for example, 1 nm to 10 nm, and more specifically, 1 nm or more and 5 nm or less, or 3 nm or more and 5 nm or less.

[0043] The diameter of ssDNA (single-stranded DNA) is about 1.5 nm, and the appropriate range of the nanopore diameter for analyzing ssDNA is about 1.5 nm to 10 nm, preferably about 1.5 nm to 2.5 nm. The diameter of dsDNA (double-stranded DNA) is about 2.6 nm, and the appropriate range of the nanopore diameter for analyzing dsDNA is about 3 nm to 10 nm, preferably about 3 nm to 5 nm. When analyzing other biopolymers, such as proteins, polypeptides, and sugar chains, the nanopore diameter can be selected according to the outer diameter of the biopolymer.

[0044] The depth (length) of the nanopore 102 can be adjusted by adjusting the film thickness of the substrate 103 or the thickness of the thin film of the substrate 103. The depth of the nanopore 102 is preferably set to the monomer unit constituting the biopolymer to be analyzed. For example, when a nucleic acid is selected as the biopolymer, the depth of the nanopore 102 is preferably set to the size of one base or less, for example, approximately 0.3 nm or less. The shape of the nanopore 102 is basically circular, but it can also be elliptical or polygonal.

[0045] At least one nanopore 102 may be provided in the substrate 103, and when multiple nanopores 102 are provided, they may be regularly arranged. The nanopores 102 may be formed by methods known in the art, for example, by irradiating with an electron beam of a transmission electron microscope (TEM), by using nanolithography techniques or ion beam lithography techniques, etc. The nanopores 102 may also be formed in the substrate by dielectric breakdown.

[0046] The chamber section 101 has a sample introduction section 104, a sample outflow section 105, a nanopore substrate 103, electrodes 114 and 115, and an electrode for passing a biological sample 113 through the nanopore 102. The chamber section 101 has a sample introduction section 104, a sample outflow section 105, a first electrode 114 provided in the sample introduction section 104, a second electrode 115 provided in the sample outflow section 105, a voltage application section 116 for applying a voltage to the first and second electrodes, and the like. An ammeter may be disposed between the first electrode 114 provided in the sample introduction section 104 and the second electrode 115 provided in the sample outflow section 105. The current between the first electrode 114 and the second electrode 115 may be appropriately determined in terms of determining the speed at which the sample passes through the nanopore. For example, when an ionic liquid not containing a sample is used, it is preferable that the current is about 100 mV to 300 mV for DNA, but is not limited to this value.

[0047] The electrodes can be made from metals, such as the platinum group metals, including platinum, palladium, rhodium, ruthenium, gold, silver, copper, aluminum, nickel, and the like; graphite, such as graphene (either single or multi-layer), tungsten, tantalum, and the like.

[0048] When a voltage is applied, the biological sample (nucleic acid molecule) 113 passing through the nanopore 102 emits Raman light due to the excitation light, but a conductive thin film may be prepared near the nanopore to generate and enhance the near field. It is also possible to improve the accuracy of base determination by taking into account not only the blockade current but also information obtained from the Raman light. The conductive thin film installed near the nanopore is formed in a planar shape, as is clear from the definition of a thin film. The thickness of the conductive thin film is 0.1 nm to 10 nm, preferably 0.1 nm to 7 nm, depending on the material used. The smaller the thickness of the conductive thin film, the more the generated near field can be limited, and analysis with high resolution and high sensitivity is possible. The size of the conductive thin film is not particularly limited, and can be appropriately selected depending on the size of the solid substrate and nanopore used, the wavelength of the excitation light used, and the like. Note that if the conductive thin film is not planar and has a bend, a near field is induced at the bend, light energy leaks out, and Raman scattering light is generated at a location outside the target. In other words, the background light increases and the S / N ratio decreases. Therefore, the conductive thin film is preferably flat, in other words, the cross-sectional shape is preferably straight without bending. Forming the conductive thin film flat is not only effective in reducing background light and increasing the S / N ratio, but is also preferable from the viewpoints of the uniformity of the thin film and reproducibility in the production.

[0049] Compartment forming portion 117 may be a part of nanopore substrate 103, may be a separate part in contact with nanopore substrate 103, or may be a part of a flow cell that houses the nanopore substrate. In the case of a biological nanopore, the same effect can be obtained by using multiple types of proteins with different diameters of the central pore or by adjusting the size of the pore by molecular modification or the like.

[0050] Fig. 4A is a diagram of the flow channel structure of a flow cell. Using the flow cell shown in Fig. 4A, the effect of differences in liquid replacement efficiency caused by the structure of the compartment formation part 117 was verified by transient analysis using three-dimensional fluid analysis software. The flow cell 418 has a flow channel 104, in which a solution flows from an inlet channel 106 to an outlet channel 108, and a nanopore substrate 103 is present midway through the flow channel 104.

[0051] Fig. 4B is an enlarged view of the nanopore substrate 103 portion of Fig. 4A. A simulation of the liquid replacement efficiency was performed when the opening size 419 of the compartment forming portion 117 was 100 nm and when it was 500 nm. The total volume of the flow channel 104 was 24 μL, the physical property of the fluid was water, the temperature of the fluid was 25°C, and the flow rate was 3 μL / s or 80 μL / s.

[0052] Figures 5A and 5B show the sections for evaluating the liquid replacement efficiency. Figure 5B is an enlarged view of the vicinity of the nanopore 102 in Figure 5A. In the flow channel 104, the section located above the nanopore substrate 103 and the section forming part 117 is section a520, and the section surrounded by the section forming part 117 directly above the nanopore substrate 103 is section b521.

[0053] 6A and 6B show the time transition of the liquid replacement rate (proportion of replacement completed) of the section a 520 and the section b 521. Fig. 6A shows the flow rate of 3 μL / s, and Fig. 6B shows the flow rate of 80 μL / s. The results of a simulation performed under the conditions that the opening size 419 of the section formation part 117 is 100 nm and 550 nm, respectively, are shown.

[0054] In FIG. 6A, at the time of 2 s when 12 μL, which is half of the flow channel volume, flows in, the liquid replacement rate of the section b 521 is about 50% for 550 nm and about 30% for 110 nm, and it can be seen that the concentration difference of about 1.7 times occurs depending on the opening size. In FIG. 6B, at the time of 0.075 s when 12 μL, which is half of the flow channel volume, flows in, the liquid replacement rate is about 95% and about 20%, respectively, and it can be seen that the concentration difference of about 4.8 times occurs depending on the opening size. From this, it was demonstrated that when nanopore substrates 103 with opening sizes 419 of 100 nm and 550 nm are mixed in a nanopore array device, the biological sample concentration difference is about 5 times due to the difference in liquid replacement rate. The opening size 419 is not limited to this numerical example, and may be various values ​​from tens of nm to tens of mm.

[0055] In Fig. 6A, it can be seen that liquid replacement begins first in section a 520, followed by section b 521, regardless of the opening size 419. On the other hand, in Fig. 6B, when the opening size 419 is 550 nm, liquid replacement progresses faster in section b 521 than in section a 520. The reason for this will be explained using Figs. 7 and 8.

[0056] 7 and 8 are schematic diagrams showing the change over time in the liquid replacement rate in section a520. FIG. 7 shows the simulation results at a flow rate of 3 μL / s, and FIG. 8 shows the results at a flow rate of 80 μL / s. In the case of a flow rate of 3 μL / s in FIG. 7, the liquid is replaced gradually from the part where the liquid flows in to the entire section. In the case of a flow rate of 80 μL / s in FIG. 8, the flow rate is large, so the flow toward the bottom of section a520 remains strong, and the liquid replacement in section b521 is completed before the entire section is replaced.

[0057] 7 and 8, the flow rate can be adjusted to adjust the liquid replacement efficiency of the compartment a 520 and the compartment b 521. Furthermore, by adjusting the opening size of the compartments in various ways, the difference in liquid replacement efficiency between the compartments can be adjusted as desired.

[0058] <First embodiment: Summary> In the biological sample analyzer 100 according to the first embodiment, the liquid replacement efficiency (liquid volume replaced per unit time) near the nanopore 102A is smaller than the liquid replacement efficiency near the nanopore 102B. This makes it possible to make the sample concentration near each nanopore 102 different for each nanopore 102. Therefore, the sample can be measured both when the sample concentration is high and when it is low, thereby widening the dynamic range of the device.

[0059] The biological sample analyzer 100 according to the first embodiment changes the liquid replacement efficiency for each compartment by changing the opening size of each compartment in the first chamber 104. This makes it possible to expand the dynamic range of the apparatus with a simple structure without increasing background noise or complicating the apparatus configuration.

[0060] <Embodiment 2> In the first embodiment, it has been described that the difference in the distance between the compartment forming parts 117 (the opening size of the compartment) creates a difference in the liquid replacement efficiency near the nanopore 102. In the second embodiment of the present disclosure, another method for creating a difference in the liquid replacement efficiency will be described.

[0061] FIG. 9A shows a configuration example near the nanopore in this embodiment. Other than the structure near the nanopore, it is the same as in embodiment 1. In FIG. 9A, the opening of the partition gradually becomes lower in a step-like manner from left to right in the drawing. Looking at a cross section of a certain place, it is as shown in FIG. 9A. As shown in FIG. 9A, the distance between the partition forming parts 117 is uniform for each partition, but the height of the partition forming parts 117 differs for each partition, so that the liquid exchange efficiency near the nanopore 102 differs for each partition. If the height of the partition forming parts 117 is high, the liquid exchange efficiency is low, and if the height is low, the liquid exchange efficiency is high. Therefore, the liquid exchange efficiency near the nanopore 102A is lower than the liquid exchange efficiency near the nanopore 102B.

[0062] In order to cause a difference in liquid replacement efficiency depending on the height of the compartment forming portion 117, it is necessary that at least a part of the side wall of the nanopore 102A compartment is higher than the side wall of the nanopore 102B compartment. The extent to which the proportion of the side wall of the nanopore 102A compartment needs to be higher than the side wall of the nanopore 102B compartment is determined by the structural correlation between the nanopore 102A compartment and the nanopore 102B compartment. For example, if there is not much difference between the high part of the side wall of the nanopore 102A compartment (the side wall located on the left side of 102A in FIG. 9A) and the side wall height of the nanopore 102B compartment, it is necessary to increase the proportion of the high part of the side wall accordingly.

[0063] 9B shows another example of the configuration near the nanopore in this embodiment. As shown in FIG. 9B, the upper surface of the compartment forming portion 117 does not have to be parallel to the nanopore substrate 103. By combining embodiments 11 and 2, a shape in which the combination of both the distance between the compartment forming portions 117 and the height of the compartment forming portions 117 varies for each compartment may be used. Whether or not the upper surface of the compartment forming portion 117 is inclined may be appropriately determined depending on, for example, ease of manufacture. The same applies to other embodiments.

[0064] In the case of bionanopores, the same effect as in this embodiment can be obtained by using multiple types of proteins with different central pore heights, using multiple types of proteins with different three-dimensional molecular structures, or adjusting the pore height by molecular modification, etc.

[0065] <Embodiment 3> In the first and second embodiments, a difference in the volume surrounded by the compartment forming portion 117 is used to create a difference in the liquid replacement efficiency in the vicinity of the nanopore 102. In the third embodiment of the present disclosure, another method for creating a difference in the liquid replacement efficiency will be described.

[0066] Fig. 10A shows an example of the configuration near the nanopore in this embodiment. Other than the structure near the nanopore, it is the same as in embodiment 1. As shown in Fig. 10A, the volume surrounded by the compartment forming portion 117 is uniform for each compartment, but the shape of the surrounded volume portion differs for each compartment, resulting in a structure in which the liquid replacement efficiency near the nanopore 102 differs for each compartment. In Fig. 10A, the compartment forming portion 117 near the nanopore 102A is perpendicular to the nanopore substrate 103, whereas it is oblique near the nanopore 102B, so the liquid replacement efficiency near the nanopore 102B is relatively lower than that near the nanopore 102A.

[0067] In Fig. 10A, the sidewall of the nanopore 102A section does not necessarily have to be perpendicular to the substrate. That is, as long as the angle of the sidewall of the nanopore 102A section to the substrate 103 is closer to 90 degrees than the sidewall of the nanopore 102B section, the same effect as in Fig. 10A can be achieved.

[0068] Fig. 10B shows another example of the configuration near the nanopore in this embodiment. In Fig. 10B, the compartment of nanopore 102A has a tapered shape tapering from nanopore 102A toward the compartment opening, and the compartment of nanopore 102B has a tapered shape tapering from the compartment opening toward nanopore 102B. This results in a relatively higher liquid replacement efficiency near nanopore 102B compared to near nanopore 102A. This is because the opening size of the compartment is larger than that of nanopore 102A.

[0069] FIG. 11A is a schematic diagram of an experimental system combining the first to third embodiments. The first to third embodiments may be combined to form a structure in which the volume and shape of the portion surrounded by the partition forming portion 117 are different for each partition. FIG. 11A is for verifying one example. The flow cell 418 has two flow paths 104 facing the nanopore substrate 103, and liquid flows through the inflow paths 106, 107 and the outflow paths 108, 109. The two flow paths 104 are filled with liquids 110, 111, and the biological sample 113 is dissolved in the liquid 110. The inflow path 107 and the outflow path 108 have electrodes 115, 114 inserted therein, and a voltage is applied by the voltage application unit 116, and the current can be measured.

[0070] Figures 11B and 11C are enlarged views showing the structures of the nanopore substrate 103 and the compartment forming portion 117 in Figure 11A. Figures 11B and C are the same objects turned upside down.

[0071] Under the experimental conditions shown in Table 1, the orientation of the nanopore substrate 103 in Fig. 11B was compared with that in Fig. 11C. In the orientation of Fig. 11B, the opening size on the side where the biological sample 113 is introduced is 550 nm, and in the orientation of Fig. 11C, the side where the biological sample 113 is introduced has an outward tapered shape and the opening size is 1032 µm. From the current value pattern measured by the electrodes 115 and 114, the frequency at which the biological sample 113 passed through the nanopore was measured.

[0072] [Table 1]

[0073] FIG. 12 shows the results of an experiment using the experimental system of FIG. 11A. When the concentration of the biological sample 113 is 5 nM, the passing frequency of the biological sample is about 100 times higher in the direction of FIG. 11C with a larger opening than in the direction of FIG. 11B with a smaller opening. For data processing, a passing frequency of 1 Hz or more is desirable, and a concentration of 100 nM or more is required in the direction of FIG. 11B, but a passing frequency of 1 Hz is obtained at 5 nM in the direction of FIG. 11C. This passing frequency is about the same as that in the case of 100 nM in the direction of FIG. 11B, and the lower limit of the concentration of the biological sample 113 that can be measured is expanded by 20 times. From the above experimental results, it was demonstrated that the difference in the volume and shape of the part surrounded by the partition forming parts 117 as shown in FIGS. 11B and C can expand the concentration range of the measurable sample. This effect is considered to be due to the combined effect of the opening size and tapered shape of the partition.

[0074] In the case of bionanopores, effects similar to those described in Figures 11A to 12 can be obtained by using multiple types of proteins with different three-dimensional molecular structures or by denaturing the three-dimensional structure around the pore by molecular modification, etc.

[0075] <Fourth embodiment> In the first to third embodiments, an example has been described in which the concentration near the nanopore 102 differs for each compartment due to the structure of the compartment forming portion. In the fourth embodiment of the present disclosure, an example will be described in which the configurations of the nanopore substrate 103 and the compartment forming portion 117 are all the same for each compartment, and the concentration near the nanopore 102 differs due to the structure of the flow channel 104 in the flow cell 418.

[0076] Fig. 13A is a top view of the flow cell in this embodiment. Fig. 13B is a three-dimensional view of the flow channel 104. Fig. 13C is an enlarged view of the periphery of the nanopore 102. The flow cell shown in Fig. 13A has 16 nanopore device compartments on the flow channel 104. Each compartment is diagrammatically illustrated by an upward-facing triangular pyramid. Liquid flows from the inlet channel 106 through the 16 nanopore compartments and then into the outlet channel 108. All 16 nanopore devices have the same configuration as shown in Fig. 11C. The other configurations are the same as those of embodiments 1 to 3.

[0077] The liquid replacement efficiency in the vicinity of the 16 nanopores shown in Figures 13A to 13C was verified by transient analysis using three-dimensional fluid analysis software. The distance 1122 between the compartment forming parts 117 was 1032 μm, the total volume of the flow path 104 from the inlet channel 106 to the outlet channel 108 was 80 μL, the physical properties of the fluid were water, the fluid temperature was 25°C, and the flow rate was 3 μL / s or 80 μL / s. The compartment for evaluating the liquid replacement efficiency was compartment c 1323 surrounded by the compartment forming parts 117 in Figure 11C.

[0078] Figures 14A and 14B show the time course of the liquid replacement rate in section c1323. Figure 14A shows the results at a flow rate of 3 μL / s, and Figure 14B shows the results at a flow rate of 80 μL / s. In both Figures 14A and 14B, it was found that the liquid replacement proceeded in the order of channel (section) 1 closest to the liquid inlet to channel 16, the furthest. Figures 14AB show that at a certain time during the replacement, the concentration difference between each channel was larger at 3 μL / s than at 80 μL / s. For example, comparing the liquid replacement rates at 15 seconds after the replacement in Figure 14A, channel 1 was 100%, channel 6 was about 85%, channel 11 was about 25%, and channel 16 was almost 0.03%, resulting in a concentration difference of more than 3000 times between the channels.

[0079] 15 is a schematic diagram showing the change over time in the liquid replacement rate of the section c1323 when the flow rate is 3 μL / s. The state of liquid replacement shown in FIG. 14A is also clear from FIG.

[0080] From the above experimental results, it was found that a concentration difference occurs between the compartments according to the relative positions of the flow channel and the nanopore device, and furthermore, the strength of the concentration gradient can be changed by the flow rate. The flow channel structure is not limited to the shape shown in Figure 13, and the shape may be changed by considering the balance between the diffusion rate within compartment c and the diffusion rate between the channels.

[0081] Fig. 16 shows another example of a configuration for forming a concentration gradient between channels. As in the structure shown in Fig. 16, by increasing the height of the partition forming part 117, the diffusion speed within the partition becomes faster than the diffusion speed between channels on the flow path 104, so that a concentration gradient can be formed between the channels. This makes it possible to achieve the same effects as those described in Figs. 13A to 15. The other configurations are the same as those of the first to third embodiments.

[0082] Increasing the distance between the nanopore substrate 103 and the flow channel 104 (which serves to connect the nanopores 102) increases the diffusion rate in the section c relative to the diffusion rate between the channels, making it possible to increase the concentration gradient. Alternatively, differences in the relative positions between the nanopore 102 and the inflow channel 106 and between the nanopore 102 and the outflow channel 108 also cause differences in the liquid replacement efficiency.

[0083] In this embodiment, the nanopore compartment is exemplified as a 4×4 array device with 16 channels, but the number and arrangement of channels are not limited to this. The channels of the nanopore device may be arranged in parallel rather than in series on the flow path, and may have a flow path structure that combines series and parallel.

[0084] In Fig. 14A, the intervals between channels on the curves of the liquid replacement rate are nearly constant, but in Fig. 14B, they are not constant, and liquid replacement is faster in channels 5, 9, and 13, which are located just before the bend in flow path 104. This is thought to be because part of the flow that hits the wall of the bend in flow path 104 is encouraged to proceed toward the inside of the compartment (i.e., perpendicularly upward to the flow path), increasing the liquid replacement efficiency of the channel located at the bend. An example of a configuration that exerts a similar effect is given below.

[0085] 17A and 17B show another example of the configuration for forming a concentration gradient between channels. As shown in FIG. 17A, by providing a protrusion 1724 near the inlet of each channel arranged on the flow path 104 in a direction that draws the flow of liquid into the compartment, the diffusion rate in the compartment c becomes larger relative to the diffusion rate between the channels, so that the concentration gradient between the channels can be made larger. On the other hand, as shown in FIG. 17B, by providing a protrusion 1724 near the inlet of each channel in a direction that obstructs the flow of liquid, the diffusion rate between the channels becomes larger relative to the diffusion rate in the compartment c, so that the concentration gradient between the channels can be made smaller. The other configurations are the same as those of the first to third embodiments.

[0086] <Fifth Preferred Embodiment> In the first to third embodiments, the partition forming portion 117 has a single layer structure. In the fifth embodiment of the present disclosure, an example will be described in which the partition forming portion 117 has a multi-layer structure, thereby forming a difference in liquid replacement efficiency between the partitions.

[0087] Fig. 18 shows an example of the configuration near the nanopore in this embodiment. The other configurations are the same as those of the first to fourth embodiments. As shown in Fig. 18, a partition forming part 1825 is further provided above the partition forming part 117, which creates a difference in liquid replacement efficiency. Specifically, the partition forming part 1825 above the nanopore 102A narrows the opening size by partially covering the opening of the partition, and the partition forming part 1825 above the nanopore 102B does not cover the opening. This results in a lower liquid replacement efficiency near the nanopore 102A than the liquid replacement efficiency near the nanopore 102B.

[0088] The partition forming portion 1825 may be formed integrally as the same member as the partition forming portion 117, or may be formed as a member separate from the partition forming portion 117. For example, it may have a structure in which a rubber sheet or the like that is easy to manufacture and process is placed over it. In the latter case, the member that creates the difference in liquid replacement efficiency as in this embodiment does not need to be in direct contact with the nanopore substrate 103.

[0089] <Sixth embodiment> FIG. 19 shows an example of the configuration near the nanopore of a biological sample analyzer 100 according to embodiment 6 of the present disclosure. The other configurations are the same as those of embodiments 1 to 5. In this embodiment, as shown in FIG. 19, the material of the partition forming portion 117 is different for each partition. By selecting a material with different wettability (hydrophilicity) for each partition, a difference in liquid replacement efficiency occurs for each partition. Furthermore, even if the material of the partition forming portion 117 is the same, the wettability may be changed for each partition by applying a surface treatment such as a coating.

[0090] If the partition forming portion 117 has high hydrophilicity, it acts to draw liquid into the partition. If the partition forming portion 117 has high hydrophobicity, it acts to direct the liquid flow in the lateral direction of the drawing (liquid flow between the partitions) in the lateral direction as it is. Therefore, it is considered that the liquid replacement efficiency of a partition with high hydrophilicity is higher than that of a partition with low hydrophilicity (high hydrophobicity). However, these effects are relative and depend on the shape and size of the partition forming portion 117, so the degree to which the hydrophilicity of each partition should be set depends on the degree to which the difference in liquid replacement efficiency between the partitions is set.

[0091] During manufacturing, nanopore substrates made of different materials are manufactured separately and then combined to realize the compartment structure according to this embodiment. In addition, in the case of a bio-type nanopore, the same effect as in this embodiment can be obtained by modifying the surface with hydrophobic or hydrophilic groups.

[0092] <Embodiment 7> FIG. 20 shows the results of a simulation of the time progression of the liquid replacement efficiency of compartment c1323 in embodiment 7 of the present disclosure. In this embodiment, differences in the liquid replacement efficiency between compartments are caused by differences in the viscosity of the liquid supplied to the compartments. In the flow path structure of FIG. 13 of embodiment 4, a comparison was made between a case where the viscosity coefficient of the liquid is equal to that of water and a case where the viscosity coefficient is four times that of water. FIG. 20 shows the results. The flow rate is 3 μL / s. The configuration of the biological sample analyzer 1 may be the same as in the above embodiments, or the structures of the compartments may be the same.

[0093] According to FIG. 20, when the viscosity coefficient is four times, the timing when the liquid replacement in the section c1323 starts to proceed becomes earlier, but the speed of the liquid replacement thereafter becomes slower. By utilizing this effect and changing the viscosity of the liquid for each nanopore, it is possible to generate a difference in the liquid replacement efficiency for each pore. Therefore, for example, if the viscosity coefficient of the liquid filled in the nanopore 102A section before supplying a sample to the nanopore 102A section is made higher than the viscosity coefficient of the liquid filled in the nanopore 102B section before supplying a sample to the nanopore 102B section, the liquid replacement efficiency near the nanopore 102A will be lower than the liquid replacement efficiency near the nanopore 102B.

[0094] As one method of changing the viscosity of the liquid for each pore, the viscosity coefficient of the preservation liquid filled in the flow path 104 may be previously gradient for each pore. When the preservation liquid is injected, a substance that increases the viscosity, such as a surfactant, is mixed, and the substance is injected into the nanopore device without being homogenized in the liquid, resulting in a concentration gradient. Alternatively, a viscosity gradient may be generated when the liquid flows in by mixing a nanopore substrate 103 with a surfactant applied to its surface with a nanopore substrate 103 without a surfactant applied thereto. As the surfactant, TWEEN (registered trademark), TritonX, etc. may be used. Alternatively, a temperature gradient may be applied by a heat source such as a heater, so that the viscosity coefficient changes depending on the temperature, resulting in a concentration gradient for each section. In either case, the biological sample analyzer 100 at least has the functions according to this embodiment.

[0095] <Modifications of the present disclosure> The present disclosure is not limited to the above-described embodiments, and includes various modified examples. For example, the above-described embodiments have been described in detail to clearly explain the present disclosure, and are not necessarily limited to those having all of the configurations described. In addition, 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. In addition, it is possible to add, delete, or replace a part of the configuration of each embodiment with another configuration.

[0096] In the above embodiment, the voltage application unit 116 may serve as a calculation unit that analyzes the biological sample (e.g., sequentially identifies base species) using the blocking current values ​​detected by the electrodes 114 and 115. In this case, a method such as (a) ignoring the blocking current of nanopores whose blocking current is less than a threshold value and using only the blocking currents of other nanopores, (b) using only the blocking current of nanopores in which a blocking current equal to or greater than a threshold value has occurred, or (c) using the blocking current values ​​from all nanopores can be used. When the blocking current values ​​from all nanopores are used regardless of the structure of the compartment, the calculation process for analyzing the biological sample is the same as in the past, and there is an advantage in that the effort of changing the calculation process can be omitted when implementing the present disclosure.

[0097] In the above embodiment, DNA is given as an example of a biological sample, but the present disclosure can also be used in devices that analyze other biological samples. That is, the present disclosure can be applied to devices that measure samples using changes in physical quantities when a biological sample passes through a nanopore. [Explanation of symbols]

[0098] 101 Observation container (chamber section) 102 Nanopore 103 Nanopore Substrate (Substrate) 104 Sample introduction section (first chamber, flow channel) 105 Sample outflow section (second chamber, flow channel) 106, 107 Inflow channel 108, 109 Outflow channel 110, 111 liquid 113 Biological specimens 116 Voltage application unit (biological sample induction unit) 114, 115 Electrode (biological sample induction part), detection part (blocking current detection part) 116 Voltage application section 117 Compartment Formation Section 418 Flow Cell 419 Distance between partition formation parts 520 Section A 521 Section B 1122 Distance between partitions 1323 Section C 1724 Protrusion 1825 Partition Formation Division

Claims

1. A biological sample analyzer for analyzing a biological sample, comprising: a substrate having first and second pores through which the biological sample passes; a first chamber and a second chamber disposed opposite to each other via the substrate; Equipped with The first chamber has a first compartment and a second compartment separated by a compartment forming portion, the first pore is disposed at a position that allows the first compartment to communicate with the second chamber; the second pore is disposed at a position that allows the second section to communicate with the second chamber, A liquid replacement efficiency when replacing the liquid in the first compartment with another liquid in a first region closer to the first pore than the opening of the first compartment is lower than a liquid replacement efficiency when replacing the liquid in the second compartment with another liquid in a second region closer to the second pore than the opening of the second compartment. A biological sample analyzing device characterized by:

2. The volume of liquid that the first compartment can accommodate is smaller than the volume of liquid that the second compartment can accommodate.

2. The biological sample analyzer according to claim 1.

3. A first opening size on a side of the first compartment not in contact with the first pore is smaller than a second opening size on a side of the second compartment not in contact with the second pore.

2. The biological sample analyzer according to claim 1.

4. The sidewall of the first compartment has a higher portion than the sidewall of the second compartment.

2. The biological sample analyzer according to claim 1.

5. The upper surface of the partition forming portion has a portion that is not parallel to the substrate.

2. The biological sample analyzer according to claim 1.

6. a sidewall of the first section has a first angle with respect to the substrate; The sidewall of the second section has a second angle with respect to the substrate that is closer to a right angle than the first angle.

2. The biological sample analyzer according to claim 1.

7. The first section has a shape that tapers from the first pore toward an opening of the first section, The second section has a shape that tapers from an opening of the second section toward the second pore.

2. The biological sample analyzer according to claim 1.

8. The biological sample analyzer further includes a flow path for supplying liquid to the first chamber, The flow path is configured to begin supplying the liquid to the second compartment before beginning to supply the liquid to the first compartment.

2. The biological sample analyzer according to claim 1.

9. the flow path has a shape in which the liquid flow is bent at least once, The second section is disposed at a corner of the bending flow path.

9. The biological sample analyzer according to claim 8.

10. The height of the compartment forming portion is configured so that the liquid diffuses faster within the first compartment or the second compartment than between the first compartment and the second compartment.

2. The biological sample analyzer according to claim 1.

11. The biological sample analyzer further comprises: A first protrusion that blocks a flow of liquid into the first compartment; or a second protrusion for promoting fluid flow into the second compartment; Equipped with at least one of the following:

2. The biological sample analyzer according to claim 1.

12. The biological sample analyzer further includes a first compartment opening that covers a portion of the opening of the first compartment, thereby making the first opening size smaller than the second opening size.

4. The biological sample analyzer according to claim 3.

13. The hydrophilicity of the sidewall of the first compartment is less than the hydrophilicity of the sidewall of the second compartment.

2. The biological sample analyzer according to claim 1.

14. The biological sample analyzer is configured so that a viscosity of a liquid filled in the first compartment before the biological sample is supplied to the first compartment is greater than a viscosity of a liquid filled in the second compartment before the biological sample is supplied to the second compartment.

2. The biological sample analyzer according to claim 1.

15. The biological sample analyzer further includes a calculation unit that analyzes the biological sample using a blocking current value generated when the biological sample passes through the pore, The calculation unit analyzes the biological sample using a current value obtained by adding up the blocking current value when the biological sample passes through the first pore and the blocking current value when the biological sample passes through the second pore.

2. The biological sample analyzer according to claim 1.

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