JP2012026986A - Nanopore-based analyzer and chamber for sample analysis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nanopore-based analyzer that performs analysis by securing a space for arranging channel ducts and an electrode and arranging a chamber having nanopores at a high density.SOLUTION: In the nanopore-based analyzer which includes a chamber 103 and a substrate 102 provided in the chamber 103 with nanopores 101 of a nanometer size provided on the substrate 102, and analyzes a sample by applying voltage to a region in the chamber 103 to make the sample pass through the nanopores 101, channel ducts 113 and 114 for making liquid flow into the chamber 103 and conductive parts 115 and 116 for applying voltage are formed integrally.

Description

  The present invention relates to an apparatus for analyzing an analysis sample such as DNA or protein through nanometer-sized pores (hereinafter referred to as nanopores), and in particular, for introducing an analysis sample introduction channel and DNA or protein through the nanopore. It relates to the electrode which applies the voltage.

  Development of methods for analyzing high molecular polymers such as DNA and proteins using nanopores called nanopores has been underway. Although it was technically difficult to open the nanopore, it was first realized in the bio field by introducing an ion channel into a lipid bilayer (Non-patent Document 1). In addition, the measurement method using nanopores was a method according to the patch clamp method used for biological ion channel measurement. Next, attempts have been made to open nanopores using a semiconductor process, and a method using an ion beam (Non-Patent Document 2) and a method using an electron beam (Non-Patent Document 3) have been developed.

As nanopores were created, methods for analyzing macromolecules such as DNA and proteins using nanopores were developed. The main technologies required for nanopore analysis are the following two.
1. Detection technology: Detects physical changes as the polymer passes through the nanopore. Movement control technology: Move polymer and pass nanopore

  The detection technology includes a blocking current method, a tunnel current method, and a capacitance method.

  The blocking current method is a method for detecting the influence of the polymer partially blocking the opening of the nanopore (Non-patent Document 4). As a specific structure, the space is separated into two by a film having nanopores, each space is filled with a liquid containing ions, and electrodes are arranged. When a constant voltage is applied to the electrode, ions move through the nanopore and current flows (ion passing current). When a charged polymer is present, the polymer is also drawn to one side by the potential difference and passes through the nanopore. At that time, since the opening of the nanopore is partially blocked, ions hardly flow, and the magnitude of the ion passing current is reduced. This is a method of analyzing the presence and components of a polymer by detecting this decrease in current value. In addition to the opening area, the difficulty of ion flow is affected by the charge state of the polymer and the interaction with the nanopore wall surface.

  The tunnel current method means that when a polymer passes through a nanopore, the tunnel current flows through a small gap between the tunnel current electrode and the polymer near the nanopore, and the presence of the polymer is detected. And a method for analyzing components (Non-Patent Documents 5 and 6).

  Capacitance method means that when the polymer passes through the nanopore, the nanopore is partially blocked, so the capacitor of the membrane having the nanopore changes, and the presence and components of the polymer are analyzed by detecting it. It is a method (nonpatent literature 7).

  The movement control technology includes a potential difference transfer method, an enzyme transfer method, and a mechanical transfer method.

  The potential difference transfer method is a method in which an electrode is placed in two spaces separated by a membrane having a nanopore and a voltage is applied to the electrode to generate an electric field. As an advantage, it can be realized with a simple structure, and no extra addition is applied to the polymer.

  The enzyme transfer method is a method in which an enzyme is arranged in the vicinity of the nanopore and the polymer is moved using a reaction between the polymer and the enzyme. For example, when the polymer is single-stranded DNA, there is a method of moving DNA one base at a time by placing a DNA polymerase near the nanopore and causing a double-stranded synthesis reaction. In this method, since the DNA needs to be located in the vicinity of the DNA polymerase, it is desirable from the viewpoint of high efficiency to use the potential difference transfer method and collect the DNA in the vicinity of the nanopore.

  The mechanical movement method is a method in which the polymer is moved by fixing the polymer to the beads and moving the beads with optical tweezers. In this method, it is necessary to put an end portion without a polymer bead into the nanopore, and at that time, a potential difference transfer method is adopted.

  Therefore, since the potential difference transfer method is used in combination with any method, the electrode that generates the potential difference is an essential component of the nanopore analyzer.

  A conventional nanopore type analyzer has two spaces across a membrane having nanopores. Each space requires an inflow path and an outflow path for introducing a sample and a solution containing ions serving as a carrier for electrical conduction. Each space requires an electrode for generating a potential difference for moving the sample and ions.

Kasianowicz J.J .; Brandin E.; Branton D.; Deamer D.W.:Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13770-13773 J.Li, D.Stein, C.McMullan, D.Brantion, M.J.Aziz, J.A.Golovchenko: 2001, Nature, 412, 166 Storm A.J .; J.H.Chen; X.S.Ling; H.Zandbergen; C.Dekker 2003, Nat. Mater. 2, 537540 D.Fologea; M.Gershow; B.Ledden; D.S.McNabb; J.A.Golovchenko; J.Li; Nano Lett 2005, 5, 10, 1905 M.Zwolak; M.D.Ventra; Nano Lett 2005, 5, 3, 421 M.Taniguchi; M.Tsutsui; K.Yokota; T.Kawai: Appl Phys Lett 95, 123701 (2009) G. Sigalov; J. Comer; G. Timp; A. Aksimentiev: Nano Lett 2008, 8, 1, 56

  As a problem, a chamber containing one or more nanopores requires a flow path and an electrode, respectively, but the arrangement space is limited, and in order to improve throughput, a plurality of chambers containing nanopores are arranged in parallel. It is difficult to increase the density.

  An object of the present invention is to provide a nanopore type analyzer and a sample analysis chamber that can secure a space for arranging flow channels and electrodes, and can perform analysis by arranging a chamber having nanopores at high density.

  In the nanopore type analyzer and the sample analysis chamber of the present invention, a portion for inflowing and / or outflowing liquid into the chamber and an electrode portion for applying a voltage to pass the sample to the nanopore are integrally formed.

  According to the present invention, the channels and electrodes necessary for nanopore analysis can be minimized, the nanopores can be arranged at high density, and the analysis can be performed at high throughput.

It is a figure which shows the outline | summary of the nanopore type | mold analyzer in the Example of this invention. It is a figure which shows the robot which changes the position of a nozzle and changes a flow path. It is a figure which shows the robot which changes the position of a nozzle and changes a flow path. It is a figure which shows the robot which changes the position of a nozzle and changes a flow path. It is a figure which shows the structure of the board | substrate in the Example of this invention. It is a figure which shows the structure in the conventional chamber as a comparative example. It is a figure which shows the structure in the chamber in the Example of this invention. It is a figure which shows operation | movement of the nanopore type | mold analyzer in the Example of this invention. It is a figure which shows operation | movement of the nanopore type | mold analyzer in the Example of this invention. It is a figure which shows operation | movement of the nanopore type | mold analyzer in the Example of this invention. It is a figure which shows operation | movement of the nanopore type | mold analyzer in the Example of this invention. It is a figure which shows operation | movement of the nanopore type | mold analyzer in the Example of this invention. It is a figure which shows the outline | summary of the nanopore type | mold analyzer in another Example of this invention. It is the figure which attracted | sucked the sample from the sample container with the voltage, and showed the state discharged to a chamber. It is the figure which showed the state which passes a nanopore, after attracting | sucking and discharging a sample with a voltage. It is the figure which showed the state which moves a sample from a chamber to an outflow path with a voltage, and wastes. FIG. 5 is a diagram showing a state in which a suction-side flow path is moved in order to suck another sample in sucking and discharging a sample by voltage. FIG. 5 is a diagram showing a state in which a sample tray is moved by a robot in order to suck another sample in sucking and discharging a sample by voltage. It is the figure which showed a mode that the sample was attracted | sucked from one of several flow paths in the suction / discharge of the sample by a voltage. FIG. 20 is a diagram illustrating a state in which a sample is sucked from one of the flow paths different from that in FIG. 19 in a plurality of flow paths in the suction / discharge of the sample by voltage. It is the figure which showed a mode that the sample was attracted | sucked simultaneously from several flow paths in the suction / discharge of the sample by a voltage. FIG. 5 is a diagram showing a state in which a back flow is prevented by a valve when a sample is sucked and discharged by voltage in a plurality of flow paths simultaneously. It is the figure which showed the state which prevented the backflow by the voltage in the case of performing simultaneously the suction / discharge of the sample by a voltage with a some flow path. It is a figure showing the state of the blocking current at the time of DNA passing a nanopore. It is a figure which shows the Example in case a plurality of chambers are arranged in parallel. It is a figure which shows another Example in case several chambers are located in parallel. It is a figure which shows another Example in case a plurality of chambers are arranged in parallel. It is a figure which shows the Example in case the sample introduction side of a chamber consists of several space. In the blocking current measurement, a structure in which the inflow and outflow paths are arranged in parallel without a partition in the chamber is shown. In the blocking current measurement, a case where there is no partition in the chamber and a structure in which the inflow paths are arranged in parallel and there is one outflow path is shown. In the tunnel current measurement, a case where there is no partition in the chamber and a structure in which inflow paths are paralleled and one outflow path is shown is shown.

  FIG. 1 shows an outline of the nanopore analyzer of this embodiment. The nanopore analyzer 100 of this example includes a chamber 103 including a substrate 102 having one or more nanopores 101, a pump mechanism 106 for feeding pure water 104 or ionic liquid 105 to the chamber 103, and a sample tray 107. The position of the nozzle 111 and the position of the nozzle 111 for connecting the flow path to the chamber 103 and the sample tray 107 are shown. And a robot 112 that changes the flow path. In addition, a part of the inflow paths 113 and 114 in the chamber 103 is conductive, and the conductive portions 115 and 116 are applied with a voltage by a power source 117, and are connected to an analog-digital converter (ADC) and data via an amplifier 118. It is connected to the logger 119 and an electric signal can be taken out. The outflow paths 120 and 121 are flow paths for allowing excess liquid in the chamber 103 to flow to waste liquid.

  Since the pump mechanism 106 only needs to feed the pure water 104 or the ionic liquid 105 in one direction, the liquid amount is not strictly controlled, and a rotary pump, a diaphragm pump, or the like is used. If different types of liquid are supplied to both sides of the substrate 102, for example, pure water is sent to one side and ionic liquid is sent to the other side, two pump mechanisms can be prepared and operated separately.

  Since the pump mechanism 109 has to suck the sample 108 with high accuracy and then discharge the sample 108, a syringe pump or a plunger pump is desirable.

  As the valves 110, 122, and 123, electromagnetic valves that open and close by electric signals are used.

  The robot 112 is preferably a θ-Z type robot driven by a cylindrical coordinate system in order to move the position of the nozzle 111. When the sample 108 is arranged on the sample tray 107 on a two-dimensional plane, for example, in the case of a plastic plate having 96 or 384 wells, the positional relationship between the nozzle 111 and the sample 108 needs to be changed two-dimensionally. Therefore, the robot 112 needs to be the θ-φ-Z type (FIG. 2) obtained by further adding one rotation joint from the θ-Z type or the XYZ type (FIG. 3). Alternatively, the nozzle 111 may be moved by a θ-Z type robot 112 and the sample tray 107 may be moved by a uniaxial robot 112 (FIG. 4).

  The power source 117 is a direct current in this embodiment, but may be a superposition of a direct current and an alternating current. In the case of direct current, an ion passing current proportional to the applied voltage and a blocking current when the polymer polymer passes through the nanopore are observed. In the case of DC and AC superposition, by detecting the phase change of the AC component, the impedance change of the nanopore can be known, and the component analysis of the polymer can be performed.

  FIG. 5 shows details of the substrate 102. A substrate 201 disposed in the chamber 103 includes a base 202, a film 203, and a coating layer 204. Regarding the material, the base 202 is made of silicon, the film 203 is made of silicon nitride or silicon oxide, or silicon carbide, and the coating layer 204 is made of silicon oxide. The film 203 has a nanometer-size hole called a nanopore 205. The size of the nanopore 205 is desirably 1 nm to 100 nm. The nanopore 205 is created by a method using Focused Ion Beam, a method using an electron beam, or the like. The film 203 may be silicon oxide. The coating layer 204 has functions of insulation, strength increase, and hydrophilicity addition, and the function can be replaced by the film 203.

  The substrate 201 shown in FIG. 5 has a film 203 at the top and a base 202 at the bottom, but when placed in the chamber 301 shown below, the upper film 203 and the lower base 202 may be reversed.

  As a comparative example, a conventional chamber 801 is shown in FIG. The chamber 801 is composed of two sealed spaces 803 and 804 with a substrate 802 therebetween, and is filled with a liquid 809 introduced through inflow paths 805 and 806 and outflow paths 807 and 808. The liquid contains a large amount of ions (hereinafter referred to as ionic liquid) that are responsible for charge and a sample having a small amount of charge to be analyzed. When a voltage is applied between the electrodes 810 and 811 arranged on both sides of the nanopore, And the sample moves through the nanopore. As the ionic liquid, an ionizable solution such as an aqueous potassium chloride solution is used.

  Next, FIG. 7 shows the chamber 312 of this embodiment. The chamber 312 is composed of two sealed spaces 314 and 315 across the substrate 313, and is filled with an ionic liquid 320 introduced through inflow paths 316 and 317 and outflow paths 318 and 319. The liquid includes a large amount of ions (hereinafter referred to as an ionic liquid) serving as a charge carrier and a sample having a small amount of charge to be analyzed, and electrodes 321 and 322 integrated with the flow path disposed on both sides of the nanopore. When a voltage is applied to, the ions and sample move through the nanopore.

  The electrodes 322 and 323 integrated with the flow path are made of, for example, a cylindrical metal (metal pipe). Metal pipes are composed of silver / silver chloride, which is commonly used in the field of electrochemistry. Or you may be comprised with stainless steel, platinum, and gold | metal | money. A part of the metal pipe may be in direct contact with the ionic liquid and energized. Therefore, it is desirable that the inner surface of the pipe is coated with tetrafluoroethylene or the like so as not to adsorb organic substances such as a polymer.

  Alternatively, a flow path having electrodes can be formed by creating a flow path by a semiconductor process, sandwiching an insulating layer, and conducting a conductive coating on the surface. For example, when the material is silicon, the flow path is formed by a semiconductor process such as anisotropic etching of silicon or a Bosch process. Next, silicon oxide is vapor-deposited on the surface as an insulating layer, and finally, gold, platinum, titanium, or the like is coated on the surface to form a channel having electrodes.

  Advantages of the integration of the electrode and the flow path include that the arrangement space can be reduced, and that the connection with the outside of the chamber can be concentrated, so that the hermeticity can be easily improved. Further, since the electrode and the inlet can be arranged at the same position, the electrode can be brought close to the nanopore, and the influence of the diffusion of the sample can be prevented and high-precision analysis is possible.

  In this device, the components of the polymer are analyzed by detecting physical changes as the polymer passes through the nanopore. For example, when the polymer is DNA, DNA is introduced into the space A via a flow path, and a voltage is applied so that the electrode in the space A is negative and the electrode in the space B is positive. Due to the potential difference, ions and DNA in the solution move. Since DNA is negatively charged, it passes through the nanopore and is attracted to the positive side. When DNA passes through the nanopore, the opening of the nanopore is blocked and the opening area is reduced, so that other ions in the liquid are difficult to flow. When a constant potential difference is applied, the difficulty of ion flow is detected as a decrease in current value. Based on the decrease, the type of DNA base and whether the DNA is single-stranded or 2 It is possible to analyze whether it is a main chain.

  Examples of the polymer for analyzing the components include DNA, RNA, and protein. In addition, single strands, double strands, and partial double strands of DNA and RNA can be detected.

  The analysis procedure will be described with reference to FIGS. The analysis procedure consists of (1) cleaning of nanopores and chamber, (2) supply of ionic fluid, (3) suction of sample, (4) discharge of sample, and (5) application / measurement of voltage.

(1) Cleaning of nanopore and chamber FIG. 8 shows a state in which pure water 104 is supplied to the chamber 103. The nozzle 111 is connected to the inflow path 113 by the robot 112. The valve 110 and the valve 122 are opened, the valve 123 is closed, the pump mechanism 106 is operated, and the pure water 104 is sent to the chamber 103. In order to remove the previously analyzed sample, a larger amount of pure water 104 than the volume of the chamber 103 is allowed to flow. The pure water 104 that cannot enter the chamber 103 is sent to the outside as waste liquid through the outflow passages 120 and 121.

(2) Supply of Ionic Liquid FIG. 9 shows a state where the ionic liquid 105 is supplied to the chamber 103. The nozzle 111 is connected to the inflow path 113 by the robot 112. The valve 110 and the valve 123 are opened, the valve 122 is closed, the pump mechanism 106 is operated, and the ionic liquid 105 is sent to the chamber 103. In order to replace the pure water 104 in the chamber 103 with the ionic liquid 105, a larger amount of the ionic liquid 105 than the capacity of the chamber 103 is allowed to flow. The ionic liquid 105 that does not enter the chamber 103 is sent to the outside as waste liquid through the outflow paths 120 and 121. (1) The nanopore and the chamber may be cleaned with the ionic liquid 105 in this step instead of pure water.

(3) Sample Aspiration As shown in FIG. 10, the sample 108 is aspirated. First, the pump mechanism 106 is stopped, then the valve 110 is closed, and then the nozzle 112 is moved to the sample position by the robot 112. Thereafter, the syringe pump 109 is pulled to suck the sample 108.

(4) Discharge of sample As shown in FIG. 11, the sample 108 is discharged. First, the nozzle 111 is moved to the chamber inflow path 113 by the robot 112, and then the syringe pump 109 is pressed to discharge the sample 108 into the chamber 103.

(5) Voltage application / measurement As shown in FIG. 12, a constant voltage is applied. For example, in the case of DNA 401 in which the high molecular polymer is negatively charged, when the conductive portion 115 is applied negatively and the conductive portion 116 is applied positively, the DNA 401 is attracted to the positive conductive portion 116 through the nanopore. At that time, the ions in the ionic liquid 105 are also attracted to the plus and minus electrodes according to the respective charges, but the holes (nanopores 101) are partially formed while the DNA passes through the nanopores 101. The current value decreases because ions are difficult to flow. The state is detected to detect the type of DNA base, whether single-stranded or double-stranded.

  When performing repeated analysis, return to (1) nanopore and chamber cleaning again, clean the inside of the chamber 103, and (3) aspirate the sample that is the same as or different from the previous one. And analyze. In the cleaning, (1) the ionic liquid 105 may be supplied instead of the cleaning of the nanopore and the chamber.

  If the chamber in the sealed space has a structure such as air vent, the inflow path and the outflow path can be made one.

  Moreover, you may provide an electrode not in an inflow path but in an outflow path. In this case, it is desirable to arrange the outflow channel with the electrode near the nanopore.

  FIG. 13 shows another flow path configuration. This structure is suitable when liquids having different components and concentrations are placed on both sides, such as when pure water 104 is placed on one side and ionic liquid 105 is placed on the other side of the substrate 102 in the chamber 103. Pump mechanisms 106 and 106 ', valves 122 and 122', and valves 123 and 123 'are arranged. When increasing the types of solutions, it is possible to handle various liquids by increasing the valves and switching each.

  FIGS. 14A and 14B show another method for introducing a sample into the chamber. The inflow path 113 having the conductive portion 115 and the flow path 125 having the conductive portion 124 are connected, and the sample 108 is transferred from the sample tray 107 to the chamber 401 by applying a voltage between the conductive portion 115 and the conductive portion 124. Moving. Specifically, when a high molecular polymer having a negative charge is moved as a sample, when a voltage higher than that of the conductive portion 124 is applied to the conductive portion 115, the high molecular polymer moves so as to be attracted to the conductive portion 115. In the method using the pump mechanism 109 of FIG. 8, there is connection / disconnection of the nozzle 111 and the inflow passage 113, but this method does not connect / disconnect the flow path, so that contamination is prevented and liquid is prevented from drying. Leads to.

  As shown in FIG. 15, the sample that has reached the conductive portion 115 is detected by passing a voltage higher than that of the conductive portion 115 to the conductive portion 116 and passing through the nanopore 101. In addition, voltage application / disconnection, voltage direction reversal, and the like are performed in accordance with the charged state of the sample and measurement conditions.

  As shown in FIG. 16, the sample can be efficiently discharged by providing a conductive portion 126 in the outflow path 120 and applying a voltage to the conductive portion 115.

  As shown in FIGS. 17A and 17B, the robot 112 can move the flow path 126 to switch the sample 108.

  Further, as shown in FIGS. 18A and 18B, the sample tray 107 can be moved by the robot 112 ′ to switch the sample 108.

  FIG. 19 shows a method for switching the sample 108 without using a robot. A conductive portion 129 is provided at the intersection of the flow channel 125 and the flow channel 128, a voltage is applied between the conductive portion 124 and the conductive portion 129, and the same potential is applied between the conductive portion 127 and the conductive portion 129. Then, the sample 108 flows only in the flow path 125 and reaches the conductive portion 129. Thereafter, in order to flow the sample to the nanopore 101, a voltage may be applied to the conductive portions 115 and 116.

  Next, as illustrated in FIG. 20, the conductive portion 124 and the conductive portion 129 are at the same potential, and a voltage is applied between the conductive portion 127 and the conductive portion 129. Then, the sample 108 flows only through the flow path 128, reaches the conductive portion 129, similarly, a voltage is applied to the conductive portions 115 and 116, and the sample flows through the nanopore 101.

  FIG. 21 shows a case where a plurality of samples are respectively introduced into a plurality of nanopores. The configuration is such that conductive parts, flow paths, and measurement systems in the case of one nanopore are arranged in parallel by the number of measurement samples. The inflow path 114 and the conductive portion 116 can be shared, but they may be arranged in parallel.

  Also, as shown in FIG. 22, by providing a valve at a location where the flow paths intersect, contamination between samples can be prevented.

  Alternatively, as shown in FIG. 23, a conductive portion 130 is provided at a location where the flow paths intersect, and a voltage is applied so that the conductive portion 130 has the same potential as the conductive portions 124 and 124 ', thereby causing contamination between samples. Can be prevented.

  FIG. 24 shows changes in the current signal when the polymer passes. In a normal state, ions flow through the nanopore, but when the polymer enters the nanopore, the ions hardly flow and the current value decreases. The component / state of the polymer is measured according to the degree of the decrease.

  FIG. 25 shows an embodiment in which a plurality of chambers 601 and 602 are arranged in parallel. Since the electrodes / channels 603 and 604 are independent of each other, separate samples can be introduced and analyzed. When the number of nozzles 111 for introducing the sample is one as shown in FIG. 1, the suction and discharge of the sample are repeated with respect to the respective chambers 601 and 602. When there are a plurality of nozzles 111, a plurality of samples can be sucked and discharged at a time. The polymer polymer component detection method can be realized by a blocking current method, and in the case of the tunnel current method and the capacitance method, it can be realized by mounting a tunnel current detection electrode or a capacitance detection electrode on the substrate 102 separately. Is possible.

  FIG. 26 shows another embodiment in which a plurality of chambers 605 and 606 are arranged in parallel. One pair of chamber and electrode / channel 607, 608 is arranged for each nanopore. Since all the channels are collected in one channel 609, one sample can be analyzed in parallel. Further, since the electrode portions are separated, voltage On / Off can be simultaneously performed for each nanopore. The component detection of the polymer can be realized by the blocking current method, and in the case of the tunnel current method and the capacitance method, it can be realized by separately mounting a tunnel current detection electrode or a capacitance detection electrode on the substrate. .

  FIG. 27 shows another embodiment in which one chamber 610 is provided and a plurality of nanopores 611 and 612 are arranged in parallel. There is one electrode / channel 613, 614, and there are a plurality of nanopores 611, 612. In the vicinity of the nanopores 611 and 612, tunnel current detection electrodes or capacitance detection electrodes 615 and 616 are arranged in the nanopores 611 and 612, respectively. Polymer component detection can be realized by a tunnel current method or a capacitance method.

  In FIG. 28, the sample introduction side of the chamber is composed of a plurality of spaces 617 and 618, nanopores 620 and 621 are arranged in each space, and one space 619 opposite to the introduction side is composed of one. Different samples can be introduced from the electrodes / channels 622 and 623, respectively, and a voltage is applied between the electrodes / channels 624. In the vicinity of the nanopores 620 and 621, electrodes 625 and 626 for detecting a tunnel current or detecting a capacitance are arranged for the nanopores 620 and 621, respectively. Polymer component detection can be realized by a tunnel current method or a capacitance method.

  As shown in FIG. 29, when the electrodes / channels 628 and 629 are arranged in the vicinity of the nanopore, diffusion from the channel to the nanopore can be suppressed to be small, and a plurality of electrodes / channels are provided in one chamber 627. It is possible to arrange and introduce different samples. Moreover, the time for the sample to reach the nanopore can be shortened.

  Further, as shown in FIG. 30, the side for introducing the sample into the chamber requires a plurality of electrodes / channels corresponding to the number of samples to be processed in parallel, but the opposite side has at least one electrode / channel 633. It is sufficient if only there is an electrode, or only an electrode may be used.

  As shown in FIG. 31, even in the case of measurement using a tunnel current or capacitance, when the electrodes and flow paths 635 and 636 are arranged in the vicinity, diffusion from the flow path to the nanopore can be suppressed to a small one chamber 634. It is possible to arrange a plurality of electrodes / channels and introduce different samples. Moreover, the time for the sample to reach the nanopore can be shortened.

  A plurality of electrodes / channels, a plurality of chambers, a plurality of nanopores, and a plurality of electrodes indicate a structure in which a plurality of elements are arranged one-dimensionally or two-dimensionally. In the drawing, each element is displayed as two pieces, but the present embodiment is not limited to this.

  It will be readily appreciated by those skilled in the art that the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the invention described in the claims.

100 Nanopore analysis apparatus 101,205,611,612,620,621 Nanopore 102,201,313 Substrate 103,312,601,602,605,606,610 Chamber 104 Pure water 105,320 Ionic liquid 106 Pump mechanism 107 Sample tray 108 Sample 110, 110 ′, 122, 123 Valve 111 Nozzle 112 Robot 113, 114, 316, 317 Inflow path 115, 116 Conductive portion 117 Power supply 118 Amplifier 119 Data logger 120, 121, 318, 319 Outflow path 124, 126, 127 , 129, 130 Conductive portions 125, 128 Channel 202 Base 203 Film 204 Coating layer 314, 315, 617, 618, 619 Space 321,322 Electrode 603, 604, 607, 608, 613, 614, 62 2,623,624 Electrode / channel 609 Channel 615,616 Measurement electrode 625,626 Electrode for capacitance detection 627,634 Chamber 628,629,633,635,636 Electrode / channel 637,638 Electrode

Claims (21)

A chamber and a substrate provided in the chamber;
The substrate is provided with nanometer-size pores,
In the nanopore type analyzer that analyzes the sample by passing the sample through the pores by applying a voltage to the region in the chamber,
A nanopore analysis apparatus, wherein a liquid flows into and / or out of the chamber from an electrode portion to which the voltage is applied. A chamber and a substrate provided in the chamber;
The substrate is provided with nanometer-size pores,
In the nanopore type analyzer that analyzes the sample by passing the sample through the pores by applying a voltage to the region in the chamber,
The nanopore type analyzer characterized in that at least a part of a portion for inflowing and / or outflowing liquid into the chamber is formed by an electrode portion for applying the voltage. A chamber and a substrate provided in the chamber;
The substrate is provided with nanometer-size pores,
In the nanopore type analyzer that analyzes the sample by passing the sample through the pores by applying a voltage to the region in the chamber,
A nanopore type analyzer characterized in that a portion for inflowing and / or outflowing liquid into the chamber and an electrode portion for applying the voltage are integrally formed. In claim 3,
A plurality of the chambers are disposed;
The nanopore type analyzer characterized in that at least any two or more of the inflow portions of the liquid into the chamber are connected on the upstream side. In claim 4,
A nanopore analyzer characterized in that a plurality of chambers share the same substrate, and the substrate is provided with pores for each chamber. In claim 3,
A plurality of the chambers are disposed;
A nanopore analyzer characterized in that a plurality of chambers share the same substrate, and the substrate is provided with pores for each chamber. In claim 3,
A nanopore analysis apparatus, wherein a plurality of the pores are provided in one chamber. In claim 7,
A nanopore type analyzer characterized in that the pores are arranged in an array. In claim 3,
At least one side separated by the substrate in the chamber is further divided into a plurality of regions, and the pore is provided in the substrate for each region. In claim 3,
The sample introduction side separated by the substrate in the chamber is divided into a plurality of regions, the pores are provided in the substrate for each region, and the other side separated by the substrate is one region. Nanopore type analyzer characterized by that. In claim 3,
A nanopore type analyzer having a pump for sucking a sample and discharging the sucked sample into the chamber. In claim 3,
A nanopore type analyzer having a dispensing mechanism that can be driven in a vertical direction and rotates about a predetermined axis. In claim 12,
The dispensing mechanism further has another rotary joint, and the nanopore type analyzer. In claim 3,
A nanopore type analyzer having a dispensing mechanism capable of being driven in the XYZ directions. In claim 3,
A nanopore type analyzer comprising a valve for switching pure water and ionic liquid to the chamber. In claim 15,
Furthermore, the nanopore type | mold analyzer provided with another valve | bulb which supplies a sample by switching. In the sample analysis chamber used for nanopore analysis,
A portion for inflowing and / or outflowing liquid, a substrate having nanometer-sized pores and partitioning an internal compartment, and an electrode for applying a voltage from both sides of the substrate to the chamber,
A sample analysis chamber, wherein the inflow and / or outflow portion and the electrode are integrally formed.   4. The nanopore analyzer according to claim 3, wherein a portion for flowing in and out of the liquid and the electrode portion are integrally formed in the vicinity of the pore.   19. The nanopore type analyzer according to claim 18, wherein at least two or more liquid inflow and outflow portions are provided in the chamber. In claim 3,
A nanopore analyzer characterized in that a sample is sent from a sample tray to a chamber by a potential difference, and is discharged from the chamber by a potential difference after measurement. In claim 3,
A nanopore type analyzer having a valve or an electrode at a portion where a plurality of flow paths intersect, and preventing mixing of a sample between the plurality of flow paths by opening / closing the valve or applying a voltage.

Images