WO2020241291A1 - Cellule d'écoulement et système de détection de réseau de nanopores - Google Patents

Cellule d'écoulement et système de détection de réseau de nanopores Download PDF

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Publication number
WO2020241291A1
WO2020241291A1 PCT/JP2020/019367 JP2020019367W WO2020241291A1 WO 2020241291 A1 WO2020241291 A1 WO 2020241291A1 JP 2020019367 W JP2020019367 W JP 2020019367W WO 2020241291 A1 WO2020241291 A1 WO 2020241291A1
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flow path
membrane
inlet
flow cell
flow
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PCT/JP2020/019367
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English (en)
Japanese (ja)
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ハイ フィ グェンファム
至 柳
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株式会社日立製作所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means

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  • the present disclosure relates to, for example, the design of a multi-channel flow cell with a plurality of channels or channels for accessing each element position within an array device.
  • Nanopores are made on a membrane sandwiched between the upper and lower chambers of an aqueous solution-filled flow cell. The electrodes are immersed in the solution in each chamber. The targeted biological sample is placed in one chamber. A potential difference is applied between the electrodes and the targeted biological sample is electrophoresed and passes through the nanopores. A targeted biological sample is detected by measuring the ionic current flowing between the electrodes. When the targeted biological sample passes through the nanopore, the ionic current is blocked depending on the structure and size of the sample.
  • Nanopore devices can be made by solid and biological methods.
  • the solid method prepares nanopores using a semiconductor material with high mechanical strength.
  • a silicon nitride (SiN) film is used as the membrane.
  • Nanometer-sized pores are made on the membrane by various methods such as irradiation with electron beam technology and dielectric breakdown technology that applies a voltage to the membrane.
  • Nanopore array sensing systems require not only pore arrays, but also adjacent components such as flow cells and electrodes.
  • Patent Document 1 discloses a multi-channel flow cell and electrodes constructed on a PCB for a nanopore array sensor. Each chamber is separated by a partition. The PCB board, nanopore array chips, and dividers are precisely glued together. After making all channels of the flow cell hydrophilic, an aqueous solution is inserted into each nanopore by laminating the liquid.
  • Non-Patent Document 1 presents another multi-channel flow cell made of an acrylic material, and each chamber is separated by an O-ring. The aqueous solution is pipette into each chamber.
  • the flow cell disclosed in the above document cannot utilize all the membranes in the membrane array chip due to the electric conductance error caused by the bubbles generated in the flow cell, and arranges a flow path to access all the membranes. There is not enough space for it.
  • the multi-channel flow cell of Non-Patent Document 1 sacrifices half of the nanopore array because there is not enough space for one inlet flow path and one outlet flow path for all the densified nanopore arrays. Need to be. Another problem with multi-channel flow cells in Patent Document 1 and Non-Patent Document 1 is that the bubbles generated between the membrane surface and the flow path also reduce the number of available nanopores in the array. Ion current cannot be measured correctly due to electrical conductance errors caused by solution leakage between.
  • the present disclosure proposes a method for designing and forming a multi-channel flow cell for a solid nanopore array to utilize all of the nanopore arrays and eliminate electrical conductance errors caused by bubble generation and solution leakage problems.
  • the present disclosure proposes the design and formation of multi-channel flow cells for solid nanopore arrays to minimize the thickness of the flow cells.
  • the present disclosure is a flow cell, in which a membrane array chip having a plurality of membranes, a common chamber, and a plurality of flow cells each connected to a common chamber via a membrane in the membrane array chip.
  • Independent chambers are provided, and a plurality of flow paths are connected to the independent chambers, and each chamber proposes a flow cell including an inlet flow path and an outlet flow path.
  • one part of the flow path is arranged in a layer parallel to the membrane array chip surface, another part of the flow path is arranged orthogonal to the membrane array chip surface, and each flow path has its own. Access to a unique membrane.
  • the number of membranes in the membrane array chip is N ⁇ M array, where M is N or less, the number of channels is N ⁇ M, and N and M are positive integers.
  • the flow path includes an inlet flow path, an outlet flow path, and first and second types of joints.
  • the present disclosure is a flow cell in which an aqueous solution is supplied to a membrane, each of which is connected to a membrane array chip having a plurality of membranes, a common chamber, and a common chamber via a membrane in the membrane array chip.
  • a flow cell with a plurality of independent chambers.
  • the inlet and outlet channels are connected to their respective independent chambers via joints to combine the inlet and outlet channels, and the joints are the inlet or outlet. It has at least one protruding flow path to which at least one of the flow paths is connected.
  • the present disclosure it is possible to utilize all the membranes of the membrane array chip and eliminate the electric conductance error caused by the generation of air bubbles and the problem of solution leakage. In addition, it is possible to minimize the thickness of the flow cell, which leads to a reduction in the cost of manufacturing the flow cell and the amount of test sample.
  • FIG. 5 is a schematic cross-sectional view of one embodiment of a common chamber. It is a schematic diagram of one embodiment of a common chamber. FIG. 5 is a schematic cross-sectional view of one embodiment of a common chamber. It is a schematic diagram of one embodiment of a common chamber. FIG. 5 is a schematic cross-sectional view of one embodiment of a common chamber. It is a schematic diagram of one embodiment of a common chamber. FIG. 5 is a schematic cross-sectional view of one embodiment of a common chamber. It is a schematic diagram of one embodiment of a common chamber. FIG. 5 is a schematic cross-sectional view of one embodiment of a common chamber.
  • the present disclosure does not limit the number of membranes in the membrane array chip, the structural arrangement of the membranes, the material of the chip, the material of the flow cell, the material of the partition, or the material of the electrode.
  • the above material is just an example.
  • N ⁇ M membrane array chips (M ⁇ N) arranged in a square, rectangular, or zigzag pattern are described.
  • FIG. 1A shows a cross-sectional view of the nanopore array sensing system.
  • 101 is a Si substrate
  • 102 is a SiN membrane
  • 103 is a poly-Si membrane or SiO2 membrane
  • 104 is a SiN membrane
  • 111 is a partition of a common chamber 113
  • 112 is an independent chamber.
  • 114 partitions, 108 and 109 are KCl aqueous solutions in the common and independent chambers, respectively, 105 and 106 are Ag / AgCl electrodes immersed in the aqueous solutions in the common and independent chambers, respectively, 107 It is an electric wire that connects the Ag / AgCl electrode to the measuring unit, and 110 and 100 are SiN membrane portions that are not sandwiched between the Si substrate 101 and the poly-Si membrane 103.
  • the measuring unit 110 can apply different voltages to the electrodes 105 and 106, and can measure the current flowing between the electrodes 105 and 106.
  • Each independent chamber 114 has electrical and solution separation from the other independent chambers.
  • the nanopores can be made on the membrane 100 based on dielectric breakdown technology by controlling the applied voltage or current using the measuring unit 110.
  • FIG. 2 shows a schematic view of the multi-channel flow cell of the nanopore array sensing system shown in FIG.
  • each independent electrode 106 and common electrode 105 are immersed in aqueous solutions 109 and 108 inside the independent flow cell 134 and the common flow cell 133, respectively, in order to be connected to the measurement unit 110.
  • FIG. 3A shows the components within a multi-channel flow cell.
  • the partition 112 of the independent chamber and the partition 111 of the common chamber are made of silicone rubber.
  • the membrane array chip 115 is sandwiched between the two partitions 111 and 112.
  • the set of the chip 115 and the partitions 111 and 112 is sandwiched between the common flow cell 133 and the independent flow cell 134.
  • Thread 116 is used to secure the position of each component within the multi-channel flow cell.
  • the common flow cell 133 includes a flow path 117 and a hole 119.
  • the independent flow cell 134 includes a flow path 120 and a hole 118. Holes 118 and 119 are used to insert screws 116.
  • FIG. 4A shows a detailed perspective view of the flow path in the independent flow cell.
  • the independent flow cell 134 includes a flow path 120 and a protrusion 121.
  • FIG. 4B shows a top view, a side view, and a front view of the independent flow cell 134.
  • 122 and 123 show typical inlet channels.
  • 124, 125, and 126 represent typical outlet channels.
  • the channels 122, 123, and 124 are arranged in different layers or at different depths to access the membrane.
  • the flow path includes an inlet flow path, an outlet flow path, and two types of joints (see FIGS. 13A and 13B), one of which connects the inlet and outlet flow paths in the same layer. The other joint, however, connects the inlet and outlet channels in different layers.
  • FIG. 5 shows a conventional arrangement in which each membrane has an inlet and outlet channels on the same layer.
  • each black circle indicates the location of the membrane within the membrane array chip, and two orthogonal solid lines indicate inlet and outlet channels on the same layer.
  • the number adjacent to each line indicates the layer number where the flow path is located. The higher the number, the deeper the layer. According to this arrangement, at least four layers are required to arrange the inlet and outlet channels for all membranes in the 4x4 membrane array chip of the conventional arrangement.
  • 6A-6D show four conventional arrangements where each membrane has an inlet and outlet channels on different layers. Solid and dotted pairs indicate inlet and outlet channels on different layers. According to this arrangement, at least four layers are required to arrange the inlet and outlet channels for all membranes in these arrangements of 4x4 membrane array chips.
  • the material cost for producing the flow cell can be reduced.
  • a more compact flow cell has the advantage of being easy to manufacture, handle, use and store.
  • the reduction in layer thickness also allows for a reduction in channel length, which can reduce the required amount of test sample, which can be valuable, rare and expensive.
  • FIG. 8A shows a conventional arrangement in which each membrane has an inlet and outlet channels on the same layer. According to this arrangement, at least three layers are required to arrange the inlet and outlet channels for all membranes in the 3x3 membrane array chip.
  • FIG. 8B shows a conventional arrangement in which each membrane has inlet / outlet and outlet channels on different layers. According to this arrangement, at least four layers are required to arrange the inlet and outlet channels for all membranes in the 3x3 membrane array chip.
  • FIG. 9A shows a conventional arrangement in which each membrane has an inlet and outlet channels on the same layer. According to this arrangement, at least five layers are required to arrange the inlet and outlet channels for all membranes in the 8x6 zigzag membrane array chip.
  • FIG. 9B shows a conventional arrangement in which each membrane has an inlet and outlet channels on different layers. According to this arrangement, at least four layers are required to arrange the inlet and outlet channels for all membranes in the 8x6 zigzag membrane array chip.
  • FIG. 9C shows a novel arrangement in which part of the membrane has inlet and outlet channels on the same layer and the other membrane has inlet and outlet channels on different layers. According to this arrangement, only three layers are required to arrange the inlet and outlet channels for all membranes in the 8x6 zigzag membrane array chip. The advantage of reducing the number of layers is the same as the arrangement of the flow path for the 4 ⁇ 4 membrane array chip described above.
  • FIG. 10A shows a conventional arrangement in which each membrane has an inlet and outlet channels on different layers. According to this arrangement, at least 6 layers are required to arrange the inlet and outlet channels for all membranes in the 9x6 membrane array chip. (Ii) Arrangement of inlet and outlet channels according to the present disclosure.
  • FIG. 10B shows a novel arrangement in which some of the membranes have inlet and outlet channels on the same layer and the other membranes have inlet and outlet channels on different layers. According to this configuration, only 5 layers are required to arrange the inlet and outlet channels for all membranes in the 9x6 membrane array chip.
  • the advantage of reducing the number of layers is the same as the arrangement of the flow path for the 4 ⁇ 4 membrane array chip described above. ⁇ Overview of the minimum number of layers required to place inlet and outlet channels to all membranes in an NxM membrane array chip>
  • Table 1 shows the minimum number of layers that need to be used to place the inlet and outlet channels to all membranes in an N ⁇ M membrane array chip (non-zigza, M ⁇ N) in three cases.
  • the inlet and outlet channels of each membrane are located on the same layer, and the type (2) the inlet and outlet channels of each membrane are located on different layers.
  • Some of the inlet and outlet channels of the membrane are arranged on the same layer, and the inlet and outlet channels of other membranes are arranged on different layers.
  • the minimum required number of layers is the multiplication of the minimum integer equal to or greater than the value obtained by dividing N by 2 and the minimum integer greater than or equal to the value obtained by dividing M by 2.
  • the minimum required number of layers is M + 1, and when M is even, the minimum required number of layers is M. If M is greater than or equal to the value obtained by dividing N by 2 and less than or equal to the minimum integer, in the case of type (3), the minimum required number of layers is M.
  • M is N or less and M is greater than the minimum integer greater than or equal to the value obtained by dividing N by 2
  • the minimum required number of layers is M-1.
  • the type (3) in which a part of the inlet and outlet channels of the membrane are arranged on the same layer and the inlet and outlet channels of other membranes are arranged on different layers is used for multi.
  • Designing a channel flow cell reduces the number of layers required to place inlet and outlet channels for all membranes in the membrane array chip, which makes the flow cell more compact. This has the advantage that the material cost for producing the flow cell can be reduced because the thickness of the flow cell is reduced.
  • a more compact flow cell has the advantage of being easy to manufacture, handle, use and store.
  • the reduction in layer thickness also allows for a reduction in channel length, which can reduce the required amount of test sample, which is usually valuable, rare and expensive.
  • FIG. 11 shows a perspective view of the configuration of the protrusion 121 of the independent flow cell in the nanopore array sensing system.
  • the distance h between the surface of the membrane 115 and the upper ends of both the inlet flow path 128 and the outlet flow path 129 closest to the membrane surface is set to be equal to or less than the general diameter d of the inlet flow path.
  • a general diameter is defined as the maximum distance between any two points on the boundary of a cross section.
  • the inserted aqueous solution 130 pushes the air in the flow path 128 or 129 outward through the outlet flow path 129 or the inlet flow path 128, and the aqueous solution is a membrane immediately after leaving the inlet flow path 128 or the outlet flow path 129. Contact the surface.
  • the aqueous solution 130 is placed inside the pool 131 to apply hydrophilic treatments such as ozone treatment, plasma treatment, ultraviolet (UV) irradiation, or chemical treatment (surface oxidation with acid) to the membrane surface. Can spread over all membrane surfaces.
  • the aqueous solution 130 moves outward via the outlet flow path 129 or the inlet flow path 128 closest to the membrane surface. Therefore, this protruding structure shown in FIG. 11 can prevent an electrical conductance error due to the generation of bubbles because the aqueous solution cannot contact the membrane surface.
  • air bubbles can be generated near the bottom region of pool 131 and therefore cannot touch the surface of membrane 115.
  • FIG. 12 shows a perspective view of another configuration of the independent flow cell protrusion 121 in the nanopore array sensing system.
  • the protrusion 121 is constructed only on the inlet flow path 128.
  • the distance from the surface (membrane surface) of the membrane 115 to the outlet flow path 127 closest to the membrane surface is set equal to the height of the pool 131.
  • the aqueous solution 130 comes into contact with the surface (membrane surface) of the membrane 115, and the hydrophilic treatment applied to the membrane surface causes the aqueous solution 130 to spread over all the membrane surfaces inside the pool 131. ..
  • the aqueous solution 130 is inside the pool 131 because the closest distance between the membrane surface and the outlet flow path 127 is much longer than the distance h between the membrane and the inlet flow path closest to the membrane surface. Air is extruded to the outside of the independent flow cell via the outlet flow path 129.
  • FIG. 13A and 13B show schematic views of two types of joints with protrusions 121 as presented above.
  • FIG. 13A shows a type (A) in which the inlet and outlet channels of each membrane are arranged on the same layer.
  • FIG. 13B shows a type (B) in which the inlet and outlet channels of each membrane are arranged on different layers.
  • the inlet channel 128 and the outlet channel 129 are arranged orthogonally (at 90 degrees) and the aqueous solution 130 is inserted through the inlet channel 128.
  • the inlet flow path 128 and the outlet flow path 129 are arranged in parallel (at 180 degrees), and the aqueous solution 130 is inserted from the inlet flow path 128.
  • the protrusions have a half moon structure to save space, maximize the flow path area of the protrusions and allow a stable structure for fabrication.
  • the inlet flow path 128 and the outlet flow path 129 can be arranged at any angle other than 180 degrees in the type (A), and can be arranged at an arbitrary angle (0 to 360 degrees) in the type (B). be able to.
  • FIG. 14 shows the configuration of the common flow cell 133, the partition 111, and the membrane array chip 115.
  • FIG. 14A shows a top view and a bottom view of the common flow cell.
  • FIG. 14B shows a cross section AA of FIG. 14A. As shown in FIG. 14B, a common location is constructed within the common chamber 137 for all membranes of the membrane array chip by the chip surface, common flow cell surface, and square hole divider 111.
  • the aqueous solution can be inserted into this common location from the flow path 135.
  • the aqueous solution then randomly flows into the membrane and exits the flow path 136.
  • the aqueous solution may be inserted from the flow path 136 into this common location.
  • the aqueous solution then randomly flows into the membrane and exits the flow path 135.
  • FIG. 15 shows another configuration of the common flow cell 133, the partition 111, and the membrane array chip 115.
  • FIG. 15A shows a top view and a bottom view of a common flow cell having this configuration.
  • FIG. 15B shows a cross section BB of FIG. 15A.
  • the flow path 138 is constructed in the common chamber 137, resulting in a membrane array chip from flow path 135 to flow path 136 or from flow path 136 to flow path 135. Access all membranes in.
  • the flow path 138 of the common chamber 137 consists of a chip surface, a common flow cell surface, and the surface of the partition 111.
  • An aqueous solution is inserted from the flow path 135 into this common location.
  • the aqueous solution then eventually flows through the flow path 138 into each membrane and exits into the flow path 136.
  • the aqueous solution is inserted from the flow path 136 into this common location.
  • the aqueous solution then eventually flows through the flow path 138 into each membrane and out into the flow path 135.
  • the generation of bubbles can be prevented by the random flow of aqueous solution on the common location of the common chamber 137.
  • the common flow cell 133 and the independent flow cell 134 described in FIG. 3A of the multi-channel flow cell can be manufactured by a 3D printer as an example.
  • the membrane array chip 115, the common partition 111, the common flow cell 133, and the independent flow cell 134 insert and tighten the screws to form a multi-channel flow cell.
  • the multi-channel flow cell can be used for nanopore applications.
  • the aqueous solution is inserted into each membrane from each corresponding inlet channel of the independent flow cell 134.
  • the aqueous solution is inserted into the common chamber 137 through the flow path of the common flow cell 133.
  • the electrode 106 is then immersed in an aqueous solution in the inlet or outlet channel of each independent chamber 114. Further, as shown in FIG. 2, the electrode 105 is immersed in the aqueous solution in the flow path of the common chamber 113.
  • nanopores of a desired size can be produced on each membrane based on dielectric breakdown technology.
  • the aqueous solution containing the sample is then inserted into the flow path of the common chamber 113 of the common flow cell 133 or the inlet flow path of each independent chamber 114 of the independent flow cell 134.
  • the movement of the sample through the nanopores can be detected simultaneously by applying a voltage to each chamber through the electrodes and measuring the ion current cutoff event.
  • the present disclosure allows the aqueous solution to be distributed independently to each membrane in the array without causing the problem of air bubbles or the problem of solution leakage between separated chambers.
  • Multi-channel flow cells for membrane array chips can be made.
  • the number of layers to build the flow cell can be minimized, which can reduce the material cost for making the flow cell, which makes the flow cell more compact. It has the advantage of being easy to manufacture, handle, use, and store, and can also reduce the required amount of test samples that can be valuable, rare, and expensive.
  • Membrane part 101 Substrate (Si) 102 Membrane (SiN) 103 Membrane (Poly-Si, SiO2) 104 Membrane (SiN) 105, 106 Electrode 107 Electric wire 108, 109, 130 Aqueous solution 110 Measuring unit 111 Partition (silicone rubber) 112 partition (silicone rubber) 113 Common chamber 114 Independent chamber 115 Membrane chip 116 Thread 117 Flow path 118, 119 Through hole 120, 122, 123, 124, 125, 126, 127 Flow path 121 Protrusion 128 Inlet flow path 129 Outlet flow path 131 Pool 133 Common flow cell 134 Independent flow cell 135, 136, 138 Flow path 137 Common chamber

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Abstract

La présente invention concerne une cellule d'écoulement multicanal pour un système de détection de réseau de nanopores qui est utilisé pour le séquençage d'ADN ou le comptage pour une utilisation de molécule cible, la cellule à flux multicanal résolvant le problème de limitation du nombre de nanopores pouvant être utilisés à l'intérieur d'une puce de réseau en raison d'un espace limité pour disposer des canaux d'écoulement, et le problème d'une erreur de conductance électrique due à la formation de bulles d'air et à une fuite de solution aqueuse. Dans la cellule d'écoulement multicanal, le nombre de membranes à l'intérieur d'une puce de réseau de membranes est de N × M réseaux, M étant inférieur ou égal à N, et le nombre de canaux d'écoulement étant N × M, N et M étant des nombres entiers positifs. En outre, les canaux d'écoulement comprennent un canal d'écoulement d'entrée, un canal d'écoulement de sortie et deux types de joints (voir FIG. 3).
PCT/JP2020/019367 2019-05-31 2020-05-14 Cellule d'écoulement et système de détection de réseau de nanopores WO2020241291A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113061531A (zh) * 2021-06-03 2021-07-02 成都齐碳科技有限公司 芯片结构、芯片组件、成膜方法、纳米孔测序装置及应用
WO2023021627A1 (fr) * 2021-08-18 2023-02-23 株式会社日立ハイテク Dispositif d'analyse d'échantillons biologiques

Citations (3)

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WO2015079510A1 (fr) * 2013-11-27 2015-06-04 株式会社日立製作所 Dispositif, procédé et trousse de mesure de courant
JP2017532562A (ja) * 2014-10-17 2017-11-02 オックスフォード ナノポール テクノロジーズ リミテッド 取り外し可能な構成要素を備えた電気デバイス
JP2018096688A (ja) * 2016-12-07 2018-06-21 株式会社日立製作所 液槽形成方法，測定装置及び分析デバイス

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015079510A1 (fr) * 2013-11-27 2015-06-04 株式会社日立製作所 Dispositif, procédé et trousse de mesure de courant
JP2017532562A (ja) * 2014-10-17 2017-11-02 オックスフォード ナノポール テクノロジーズ リミテッド 取り外し可能な構成要素を備えた電気デバイス
JP2018096688A (ja) * 2016-12-07 2018-06-21 株式会社日立製作所 液槽形成方法，測定装置及び分析デバイス

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113061531A (zh) * 2021-06-03 2021-07-02 成都齐碳科技有限公司 芯片结构、芯片组件、成膜方法、纳米孔测序装置及应用
WO2023021627A1 (fr) * 2021-08-18 2023-02-23 株式会社日立ハイテク Dispositif d'analyse d'échantillons biologiques
GB2623005A (en) * 2021-08-18 2024-04-03 Hitachi High Tech Corp Biological sample analysis device

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