Biomolecule detection device and biomolecule measurement method

Abstract

To provide a technique for detecting biomolecules by releasing minute amounts of biomolecules into a region where the biomolecules can be effectively drawn in by electrical attraction from nanopores in order to lower a detectable concentration.SOLUTION: A biomolecule detection device provided herein comprises a laser element of a wavelength that generates heat in a nanomembrane and magnetic beads, and is configured to position the magnetic beads bound to biomolecules on the nanomembrane surface using magnetic force, then place the magnetic beads in a range near nanopores where electrical attraction acts, heat the magnetic beads with a laser to separate the biomolecules, and detect changes in an electrical signal when the biomolecules separated from the magnetic beads pass through the nanopores.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a biomolecule detection device and a biomolecule measurement method. [Background technology]

[0002] A technology using nanopores is known as a means of detecting biomolecules such as proteins and nucleic acid molecules present in aqueous solution. This technology involves creating nano-sized holes (called "nanopores") in a thin membrane, similar in size to the biomolecules, placing the biomolecules to be detected on one side of an aqueous solution separated by this thin membrane, and measuring the change in the electrical signal generated between electrodes placed in the aqueous solution on both sides to detect the biomolecules.

[0003] In this technology, the detectable biomolecule concentration is affected by the means used to pass the biomolecules through the nanopore. In other words, if biomolecules are simply placed in an aqueous solution, the only way to increase the probability that the biomolecules will be present near the nanopore is to increase the biomolecular concentration. To address this issue, Patent Document 1, for example, discloses a technique for transporting biomolecules near a nanopore using an immobilization member to which the biomolecules are immobilized, thereby controlling the density so that at least one biomolecule enters the electric field region around the nanopore. Patent Document 1 also discloses that biomolecules can be introduced into a nanopore without confirming the nanopore's position within the thin film. Thus, in conventional biomolecule detection techniques using nanopores, the biomolecule concentration required to be detectable was such that biomolecules transported to an approximate position within the thin film could stochastically pass through the nanopore. Specifically, to effectively detect biomolecules, a detection concentration of several micromolar to several nanomolar was required.

[0004] Incidentally, the present inventor has disclosed a nanopore formation technology in Patent Document 2. This technology involves forming a nanopore directly in a thin film by laser etching. Using this technology, it is possible to determine the location of the nanopore in the thin film. The present inventor then discovered that by accurately transporting a biomolecule to a predetermined location and passing it through the nanopore, the number of biomolecules that would be stochastically required becomes unnecessary, making it possible to detect even very small amounts of biomolecules, leading to the present invention. Note that in this specification, laser light will simply be referred to as a "laser." [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2018-087819 [Patent Document 2] US Patent Application Publication No. 2019 / 0302619 Summary of the Invention [Problem to be solved by the invention]

[0006] As mentioned above, in conventional biomolecule detection devices equipped with a nanomembrane having a nanopore, in order to obtain biomolecules that pass through the nanopore, it was necessary to have excess molecules that do not pass through the nanopore also present in the liquid tank, which resulted in a problem of high detectable concentrations.

[0007] The present invention has been made in consideration of the above-mentioned problems, and aims to provide a technology that enables detection and measurement of biomolecules by releasing minute amounts of biomolecules within an area where the biomolecules can be effectively attracted by electrical attraction from a nanopore, in order to lower the detectable concentration. [Means for solving the problem]

[0008] As a result of extensive research, the present inventors have found that the above problems can be solved by providing the following configuration, and have thus completed the present invention.

[0009] (1) A biomolecule detection device comprising a flow path cell, a measurement unit, a light irradiation unit, and a magnet, wherein the flow path cell comprises a first liquid tank containing a first electrolyte solution, a second liquid tank containing a second electrolyte solution, and a nanomembrane having a nanopore separating the first and second liquid tanks, and the first liquid tank contains magnetic beads to which biomolecules are bound, the light irradiation unit comprises a laser element that emits a laser of a wavelength that is absorbed by the nanomembrane and the magnetic beads to generate heat, the magnet is positioned outside the flow path cell, and the measurement unit has the function of detecting a change in an electrical signal generated when the biomolecules separated from the magnetic beads move from the first liquid tank through the nanopore completely into the second liquid tank. (2) The biomolecule detection device described in (1) is characterized in that the light irradiation unit has the function of controlling the laser output density so that it is smaller when separating biomolecules from magnetic beads than when forming a nanopore in a nanomembrane, and has the function of being able to move the laser irradiation position within the range where electrical attraction acts near the nanopore. (3) The biomolecule detection device described in (1) is characterized in that the light irradiation unit has a feedback control function that controls the output density of the laser according to the measurement results of the measurement unit, and the magnetic force from the magnet acts to bring the magnetic beads into contact with or close to the first liquid tank surface of the nanomembrane when separating biomolecules from the magnetic beads, and does not act on the magnetic beads when forming a nanopore in the nanomembrane. (4) A biomolecule detection device comprising a flow path cell, a measurement unit, a light irradiation unit, and a magnet, the method comprising the steps of: placing magnetic beads in contact with or close to the surface of a first liquid tank of a nanomembrane by the magnetic force of a magnet placed outside the flow path cell; placing the magnetic beads in a range where biomolecules are separated by a laser emitted from the light irradiation unit; heating at least one of the magnetic beads and the nanomembrane with which the magnetic beads are in contact or close by by the laser emitted from the light irradiation unit to separate the biomolecules; and moving the separated biomolecules from the first liquid tank completely through a nanopore into a second liquid tank, wherein the range where biomolecules are separated is a range where electrical attraction acts near the nanopore, and the biomolecules are detected by detecting a change in the electrical signal using the measurement unit. (5) A biomolecule measurement method according to (4), characterized in that it includes a step of laser etching a nanomembrane placed in a flow cell with a laser emitted from a light irradiation unit to form a nanopore. [Effects of the Invention]

[0010] As described above, the present invention provides a technology that enables detection and measurement of biomolecules by releasing minute amounts of biomolecules within a region where the biomolecules can be effectively drawn in by electrical attraction from a nanopore. [Brief explanation of the drawings]

[0011] [Figure 1] 1A and 1B are diagrams showing an example of a configuration of a biomolecule detection device according to the present invention. [Figure 2] FIG. 1 is a conceptual diagram relating to capture distance. [Figure 3] FIG. 1 is a diagram showing steps for measuring biomolecules. [Figure 4] FIG. 1 is a conceptual diagram of step (S2) of placing magnetic beads on the nano-thin film surface. [Figure 5] FIG. 10 is a conceptual diagram of the step (S3) of placing magnetic beads within the capture distance range. [Figure 6] FIG. 1 is a conceptual diagram of the step (S4) of eluting biomolecules from magnetic beads. [Figure 7] FIG. 1 is a schematic diagram showing an enlarged view of the vicinity of the nanopore in step (S4) of eluting biomolecules from magnetic beads. [Figure 8] 10A and 10B are conceptual diagrams of the inside of a flow channel cell in step (S5) of measuring biomolecules passing through a nanopore, and an example of measurement results. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, a mode for carrying out the present invention (hereinafter referred to as "embodiment") will be described in detail with reference to the drawings. Note that the present invention is not limited to the specific examples described in this embodiment.

[0013] (Biomolecular detection device) The biomolecule detecting device 100 in this embodiment includes at least a flow path cell 101, a measurement unit, a light irradiation unit, and a magnet 108. In addition, the biomolecule detecting device may include a flow path cell setting stage 109 (FIG. 1).

[0014] (flow cell) The flow path cell 101 includes at least a liquid reservoir and a nano-membrane 104. In addition, the flow path cell may include an infusion pump, a temperature control mechanism, a solution stirring mechanism, and the like (not shown).

[0015] (Liquid tank: configuration) The liquid reservoirs consist of a first liquid reservoir containing a first electrolyte solution 102 and a second liquid reservoir containing a second electrolyte solution 202. In addition to the first and second liquid reservoirs, a liquid reservoir for a reference electrode, a liquid reservoir for sample preparation, a liquid reservoir for replacing the electrolyte solution, and the like (not shown) may also be provided.

[0016] (Liquid tank: electrolyte solution) The first electrolyte solution 102 and the second electrolyte solution 202 placed in the liquid tank may be any aqueous solution capable of detecting changes in the electrical signal between the electrodes in the measurement unit described below, such as KCl, NaCl, LiCl, CaCl2, or MgCl2. A buffer solution may also be used to stabilize biomolecules, such as HEPES, Tris, MOPS acetate, or citrate. The electrolyte solution may also contain a surfactant, glycerin, an organic solvent, polyethylene glycol, or a biological fluid such as serum. The electrolyte solution 102 entering the first liquid tank and the electrolyte solution 202 entering the second liquid tank may be the same, or they may be different solutions, such as aqueous solutions or buffer solutions of the same type and concentration. If the first and second electrolyte solutions are not the same, it is possible to control the three-dimensional shape of the nanopore by changing the etching rate of the surface of the nanomembrane that contacts the first liquid tank and the surface that contacts the second liquid tank, and it is possible to change the nanopore passage characteristics and capture characteristics of the biomolecule to be detected. Therefore, it is preferable for measurements that the electrolyte solutions entering the first liquid tank and the second liquid tank be matched to the biomolecule to be detected.

[0017] (Liquid tank: material) The material used to make the liquid tank is preferably a resin material with high heat and chemical resistance. Using such a material to make the liquid tank can prevent the liquid tank from being degraded by heat or chemicals. Other materials suitable for processing the flow path include Si, SiO2, and PMMA.

[0018] (nano thin film) The flow path cell is provided with a nanomembrane 104 having a nanopore that separates a first liquid reservoir from a second liquid reservoir.

[0019] (Nano thin film: material) The nano-thin film 104 is, for example, SiN x (x is greater than 0 and less than 2) can be used. x The use of such a material is advantageous in that it has high chemical resistance, corrosion resistance, thermal shock resistance, and oxidation resistance. Other materials that can be used include Si, HfO2, SiO2, MoS2, and SiC.

[0020] (Nano thin film: structure) The thickness of the nano-thin film 104 is preferably 0.1 nm to 999 nm, more preferably 0.1 nm to 100 nm, and even more preferably 0.1 nm to 50 nm. This thickness affects the capture distance of biomolecules, which will be described later, and reducing the thickness has the advantage of extending the capture distance.

[0021] The nano-thin film may be formed on a SiN substrate directly, or on a Si / SiO2 / SiN xA laminated structure as shown in the figure may be formed. In this example of the laminated structure, Si functions as a substrate 121 for holding a nano-thin film having the above-mentioned thickness in the flow path cell. SiO2, which forms the intermediate layer 122, has low capacitance and can reduce current noise. In this way, the first liquid tank and the second liquid tank are separated by a nano-thin film having a structure that can hold the film.

[0022] (Nanopore: Etching) The nanopore 105 may be formed by focused ion beam processing, focused electron beam processing, wet etching, or dielectric breakdown, but is preferably formed by laser etching. For laser etching, the technology disclosed in Patent Document 2 can be used, for example. Forming the nanopore directly in the nanomembrane provided in the flow path cell has the advantage of being able to form a single nanopore at the desired position. The laser etching is preferably performed by controlling the laser power density, for example, by adjusting the laser voltage through feedback control in accordance with the ion current measurement results obtained by a measurement unit (described later).

[0023] (Nanopore: pore size) The nanopore size can also be within the range disclosed in Patent Document 2, but in this embodiment it is preferably 0.1 nm to 999 nm, more preferably 0.1 nm to 50 nm, and even more preferably 0.1 nm to 10 nm. This size affects the detection sensitivity of biomolecules, and reducing the pore size has the advantage of enabling high-sensitivity detection of biomolecules. Note that, since the nanopore shape formed by laser etching is conical or hourglass-shaped, the pore size refers to the size at the smallest value within the thickness of the nanomembrane 104.

[0024] (nanopore: capture distance) Figure 2 is a conceptual diagram of the capture distance R of a biomolecule suitable for biomolecule detection. The potential V(r) spreading around the nanopore at a distance r from the nanopore has the following relationship among the distance r from the nanopore, the nanopore diameter d, the nanopore length l, and the voltage V applied between the first electrode and the second electrode described below:

[0025] V(r)=d 2 / 8l×1 / r×V (1)

[0026] In this equation (1), based on the relationship between the diffusion coefficient D and the electrophoretic mobility m of a biomolecule, the range of potentials where D / m or more is approximately the capture distance R of the biomolecule. This is due to the competition between the electrophoresis phenomenon, in which the charge of the biomolecule itself acts as an electrical attraction and the biomolecule is drawn to the nanopore when it enters this potential range, and the free diffusion phenomenon of the biomolecule.

[0027] In addition, when the nanopore has a conical or hourglass shape, the nanopore length is not necessarily equal to the thickness of the nanomembrane, but is the effective length that contributes to the ionic current when biomolecules or ions pass through. To improve detection sensitivity, it is preferable to adjust the nanopore diameter, nanopore length, etc. depending on the biomolecule to be detected.

[0028] (Measurement part) The measurement unit includes at least a first electrode 103b in contact with the first electrolyte solution, a second electrode 103a in contact with the second electrolyte solution, a personal computer 110, an ammeter 111, a power supply 112, a nanopore detection function, and a biomolecule detection function. The first and second electrodes are connected to the ammeter and the power supply, and a voltage is applied between the electrodes by the power supply. The application of voltage by the power supply is controlled by the personal computer, and this configuration provides the measurement unit with at least a nanopore detection function and a biomolecule detection function.

[0029] (Measurement part: electrode) In this embodiment, the electrodes are not limited in material or shape as long as they can detect the ionic current inside the flow cell via the nanopore, but Ag / AgCl electrodes are particularly preferred. Alternatively, platinum electrodes, gold electrodes, glassy carbon electrodes, silver electrodes, carbon paste electrodes, nickel electrodes, palladium electrodes, etc. can be used for the first and second electrodes.

[0030] (Measurement unit: PC) In this embodiment, the personal computer 110 refers to an electronic computer and includes an information processing device equipped with an electronic computer. The information processing device may be a so-called notebook computer or desktop computer, or may be a highly portable mobile information terminal such as a so-called smartphone or tablet terminal. The personal computer may be connected via a telecommunications line (a so-called network).

[0031] (Measurement part: ammeter) The ammeter 111 measures the ion current flowing between the first electrode and the second electrode. The value of the ion current is input to a personal computer. In this case, an amplifier for amplifying the value measured by the ammeter, an analog / digital converter, etc. (not shown) may be provided between the ammeter and the personal computer.

[0032] (Measuring part: power supply) There are no limitations on the power supply 112 as long as it is compatible with the electrodes used in this embodiment, but when Ag / AgCl electrodes are used in particular, a constant voltage power supply or a function generator can be used.

[0033] (Measurement unit: nanopore detection function) The measurement unit of this embodiment preferably has a nanopore detection function. When forming a nanopore in a nanomembrane incorporated in a flow path cell by laser etching, the nanomembrane is etched while adjusting the laser intensity and voltage by feedback control, and the nanopore is detected by changes in the ion current value. In particular, when the nanomembrane is held in a laminated structure, it is possible to finally form a nanopore in the nanomembrane by etching each of the layers of different materials appropriately.

[0034] (Measurement unit: biomolecule detection function) The measurement unit of this embodiment preferably has a biomolecule detection function. The biomolecule detection function detects changes in the electrical signal generated when a biomolecule 107 separated from a magnetic bead 106 moves from the first liquid tank to the second liquid tank by free diffusion, completely passing through the nanopore 105. A biomolecule present within the capture distance of the nanopore in the first liquid tank is attracted to the nanopore by electrical attraction, enters the nanopore, completely passes through the nanopore, and then moves into the second liquid tank by free diffusion, escaping the potential prevailing around the nanopore on the second liquid tank side. During this movement from the first liquid tank to the second liquid tank, the ionic current between the electrodes decreases from the time the biomolecule enters the nanopore until it completely passes through. Biomolecules can be detected by measuring this change in ionic current. Detecting such changes in the electrical signal is desirable for biomolecule detection.

[0035] In addition to ion current, detection is also possible using, for example, fluorescence emitted by an ion indicator. Specifically, a buffer containing ions that the ion indicator reacts to (binds to) is added to the cis side, and the ion indicator is added to the trans side. An optical window is placed on the bottom surface, and light of a wavelength absorbed by the ion indicator is irradiated. In this case, excitation light can be irradiated near the nanopore, or light can be irradiated over a wide area across the entire thin film. When the ions that the ion indicator reacts to (binds to) move electrically from the cis side to the trans side and bind to the ion indicator, they react with the excitation light and emit fluorescence. Biomolecules can be detected in this way.

[0036] (Light irradiation part) The light irradiation unit includes at least a laser element 114 , a laser driver 113 , a shutter 115 , a mirror 116 , and an objective lens 117 .

[0037] (Light irradiation part: laser element) The light irradiation unit preferably includes a laser element 114 that emits a laser with a wavelength that is absorbed by the nanothin film 104 and magnetic beads 106 and generates heat. By using a laser element with a wavelength that can be absorbed by both the nanothin film and the magnetic beads, etching of the nanothin film and separation of biomolecules from the magnetic beads to which the biomolecules are bound can be achieved using the same optical system. Using the same optical system allows the position of the nanopore in the nanothin film to be accurately determined to within a micrometer, thereby accurately determining the capture distance range of several micrometers from the nanopore. By moving the magnetic beads within this capture distance range and then releasing the biomolecules, it is theoretically possible to detect trace amounts of biomolecules, such as only those bound to a single magnetic bead, which is advantageous.

[0038] In this embodiment, the laser wavelength is preferably 10 nm to 700 nm, more preferably 380 nm to 700 nm, and even more preferably 532 nm. This wavelength range is advantageous because it allows both etching of nano-thin films and elution of biomolecules from magnetic beads to be performed with the same laser element.

[0039] (Light irradiation unit: laser driver) The laser driver 113 is not particularly limited and may be any type that can drive the above-mentioned laser element 114 and generate the power density required for laser etching of nano-thin films, movement of magnetic beads, and elution of biomolecules from magnetic beads.

[0040] (Light irradiation part: laser output density) For etching of nano-thin films, for example, as disclosed in Patent Document 2, when a laser with a wavelength of 10 nm or more and 700 nm or less is used, 5 ~10 8 W / cm 2 A laser driver capable of generating an average power density of 10 is preferred. 5 ~10 7 W / cm 2A driver capable of generating an average power density of 10 is preferred, and for eluting biomolecules from magnetic beads, 10 2 ~10 5 W / cm 2 A driver capable of generating an average power density of 1000 kJ / s is preferred. A preferred laser driver for use in this embodiment is capable of outputting all of the power densities described above (steps), and as described above, is also preferably capable of controlling the laser power density so that it is lower when separating or eluting biomolecules from magnetic beads than when forming nanopores in the nanomembrane. The power density can be adjusted by changing the laser focusing area using an objective lens, or by directly adjusting the laser output emitted from the laser driver. Any means can be used as long as it can adjust the laser power density received by the nanomembrane surface or magnetic beads. Furthermore, it is even more preferable if the driver has the function of supporting feedback control, which controls the laser power density according to the measurement results of the voltage of the measurement unit during etching. Laser manipulation does not necessarily require voltage application to the measurement unit and is possible without feedback control, but feedback control may be provided when automating biomolecule measurement, etc.

[0041] (Light irradiation part: optical element) In this embodiment, the optical system includes at least the optical elements of a shutter 115, a mirror 116, and an objective lens 117. The optical system configured with these optical elements enables the system to have a function of moving the laser irradiation position within the range where the magnetic beads are positioned when separating biomolecules from the magnetic beads and where an electric attractive force acts near the nanopore. The optical system in this embodiment has a configuration in which, for example, a laser emitted from a laser element passes through a shutter, is reflected by a mirror, passes through an objective lens, and is irradiated onto a nano-thin film or magnetic beads. In this case, the shutter functions to adjust the power density, and the mirror functions to adjust the optical path of the laser. The laser irradiated onto the nano-thin film or magnetic beads is focused by the objective lens, and the irradiation area and power density are adjusted. The optical element in this embodiment may be made of any material and have any shape as long as it has the above-mentioned functions.

[0042] (magnet) The magnet in this embodiment functions to bring the magnetic beads into contact with or close to the surface of the nanomembrane facing the first liquid layer. The magnet is preferably disposed outside the flow cell, and more preferably, the magnet is disposed outside the flow cell on the second liquid tank side of the nanomembrane. For example, if the magnet is disposed in the second liquid tank, it can bring the magnetic beads into contact with or close to the surface of the nanomembrane facing the first liquid layer. However, since this can impede the movement of the biomolecules to be detected from the first liquid tank through the nanopore into the second liquid tank, it is preferable to dispose the magnet outside the flow cell. Furthermore, the magnet may be any type that generates magnetic force, and other than a permanent magnet, it may be, for example, a magnet that generates magnetic force by passing a current through a coil.

[0043] When a nanopore is formed in a flow channel cell by laser etching from the side of the first liquid tank containing magnetic beads, it is advantageous for detecting trace amounts of biomolecules to prevent the magnetic beads from coming into contact with or close to the surface of the nanomembrane facing the first liquid layer during laser etching. Therefore, it is preferable to position the magnet so that the magnetic force from the magnet acts to bring the magnetic beads into contact with or close to the surface of the nanomembrane in the first liquid tank when separating biomolecules from the magnetic beads, but does not act on the magnetic beads when forming a nanopore in the nanomembrane. The magnetic force can be adjusted by physically changing the distance between the magnet and the magnetic beads, or it can be adjusted electrically. Alternatively, the magnetic beads can be added to the first liquid tank after forming the nanopore.

[0044] (Flow path cell installation stage) In this embodiment, a flow path cell installation stage 109 for installing the flow path cell may be provided. In this case, it is preferable to use a laser-transmitting cover glass 123 between the first liquid tank and the flow path cell installation stage for the flow path cell. By driving the flow path cell installation stage, the position of the flow path cell relative to the laser irradiation position can be changed, and the laser irradiation position on the nanothin film surface or the magnetic beads can be moved to a position when separating biomolecules from the magnetic beads, within the range where electrical attractive force acts near the nanopore.

[0045] (magnetic beads) In this embodiment, the biomolecule detection device 100 includes magnetic beads 106 in a first liquid tank, each having a biomolecule 107 bound thereto. Magnetic beads are widely used for screening and sorting biomolecules. They are particles with a magnetic core and a site, such as a linker, that binds to the biomolecule to be detected. In this embodiment, the magnetic beads 106 can be positioned by a magnet in contact with or close to the nanofilm surface facing the first liquid layer, and can be moved within a capture distance range near the nanopore by a laser from the light irradiator. As a result, when separating biomolecules from the magnetic beads, the magnetic beads can be positioned within a range where electrical attraction acts near the nanopore.

[0046] (biomolecules) The biomolecule 107 in this embodiment may be naturally occurring, synthetic, or a derivative that does not exist in nature. Specific examples of biopolymers include nucleic acids, such as single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), and hybrid nucleic acids composed of DNA and RNA; peptide nucleic acids; proteins, polypeptides (including peptides of 100 mers or shorter), such as proteins and polypeptides composed of D- or L-amino acids; and sugar chains, such as sugar chains of polysaccharides and glycoproteins, but are not limited to these.

[0047] (Elution from magnetic beads) Biomolecules 107 bound to magnetic beads 106 can be detached from the magnetic beads and eluted by heat. If the eluted biomolecules have a three-dimensional structure, such as a protein, heat may cause the three-dimensional structure to change from its native state. However, this does not significantly affect the electrical signal passing through the nanopore, allowing detection in this embodiment. While infrared rays or microwaves with wavelengths of 800 nm or longer (e.g., as described in Japanese Patent No. 7284470) are often used to heat magnetic beads, even lasers with wavelengths of 700 nm or shorter, which are preferred in this embodiment, are known to absorb a few percent of light from the magnetic material constituting the magnetic beads, and approximately 10% of light near 600 nm. Considering that magnetic beads have micrometer-sized diameters and do not emit fluorescence, most of the energy absorbed from light is converted into heat, in this embodiment, a light irradiation unit with a suitable laser power density for eluting biomolecules from magnetic beads can provide sufficient heat to the magnetic beads for elution of the biomolecules.

[0048] The configuration of the above-described embodiment makes it possible to lower the detectable concentration of biomolecules, and provides a technology that allows for the release of minute amounts of biomolecules into an area where the biomolecules can be effectively drawn in by electrical attraction from the nanopore required for biomolecular detection, thereby enabling detection and measurement.

[0049] Next, the steps of detecting biomolecules in this embodiment will be described in more detail with reference to FIGS.

[0050] (Measurement method) In this embodiment, the method for detecting a biomolecule includes at least the following steps: By performing these steps, it is possible to detect a trace amount of a biomolecule.

[0051] As shown in Figure 3, the biomolecule measurement method using this embodiment includes the steps of forming a nanopore (S1), placing magnetic beads bound to biomolecules on the nanofilm surface (S2), placing the magnetic beads bound to biomolecules within the capture distance range (S3), eluting the biomolecules from the magnetic beads (S4), and measuring the change in the electrical signal when the eluted biomolecules pass through the nanopore (S5).

[0052] The biomolecule measurement method using this embodiment will be described in more detail below. It includes the steps of (S1) laser etching the nanomembrane with a laser emitted from at least a light irradiator to form a nanopore, (S2) placing magnetic beads in contact with or adjacent to the surface of the nanomembrane in the first liquid tank by the magnetic force of a magnet placed outside the flow channel cell, (S3) placing the magnetic beads in an area where the biomolecules will be separated by the laser emitted from the light irradiator, (S4) heating at least one of the magnetic beads and the nanomembrane with which the magnetic beads are in contact or adjacent with the laser emitted from the light irradiator to separate the biomolecules and elute them into the first electrolyte solution, and (S5) moving the eluted biomolecules from the first liquid tank completely through the nanopore by free diffusion into the second liquid tank. Each step is described in more detail below.

[0053] (S1: Nanopore formation step) First, a nanopore is formed in a nanomembrane placed in a flow cell where no nanopore is formed, using, for example, the laser etching technique disclosed in Patent Document 2. When forming the nanopore, it is preferable to form a nanopore with the desired capture distance by detecting the change in ion current that occurs during nanopore formation using a nanopore detection function. Once the nanopore is formed, the nanopore position is recorded. The position can be recorded using a personal computer in the measurement unit, by storing the membrane shape based on an optical image, by recording setting information for the optical elements that make up the optical system, or by recording position information or stage drive information for the flow cell installation stage. Recording the nanopore position is related to step S3, which will be described later.

[0054] (S2: Placement on the nano-thin film surface) Next, the magnetic force is adjusted so that the magnetic beads are in contact with or close to the nanothin membrane surface facing the first liquid layer. The magnetic force can be adjusted either physically or electrically, as long as the magnetic beads can be positioned near the nanothin membrane surface using a magnet. Figure 4 shows an example of adjusting the magnetic force by narrowing the distance between the magnet placed on the second liquid tank side of the nanothin membrane and the nanothin membrane. This step allows the magnetic beads to be efficiently positioned near the nanothin membrane surface.

[0055] (S3: Placement step within capture distance range) Next, the magnetic beads positioned near the nanofilm surface are moved within the range of the nanopore where electrical attraction acts. Preferably, the magnetic beads are positioned within 100 μm of the nanopore center. This magnetic bead movement can be achieved using, for example, the laser manipulation technology disclosed in U.S. Patent Application Publication No. 2019 / 0113453. The magnetic bead's destination is preferably within the capture distance from the nanopore position recorded in step S1. This embodiment, which allows laser etching and laser manipulation to be performed using the same optical system, can move the magnetic beads with positional accuracy on the order of micrometers or less in a short period of time. The magnetic beads are moved to the range where biomolecules are eluted in step S4, i.e., the range where electrical attraction acts near the nanopore. During this magnetic bead movement, it is preferable to maintain the magnetic force applied in step S2. As described above, the biomolecule detection device of this embodiment can perform laser etching and laser manipulation using the same optical system, and when placing magnetic beads with positional accuracy of less than a micrometer, it is possible to confirm the position of the nanopore and the position of the magnetic beads to be moved by laser manipulation in a short time, which is advantageous for biomolecule measurement throughput.

[0056] (S4: Biomolecule elution step) The magnetic beads moved within the capture distance are irradiated with a laser from the same optical system used for laser etching and laser manipulation, heating the magnetic beads and separating the biomolecules bound to the magnetic beads from the magnetic beads and eluting them into the first electrolyte solution. If the magnetic force applied in step S2 is maintained, the magnetic beads are positioned near the nanothin film surface, and the area on the nanothin film surface irradiated with the laser is also heated, generating heat that contributes to the elution of the biomolecules. This embodiment thus uses a laser element with a wavelength that can be absorbed by both the nanothin film and the magnetic beads. Therefore, the biomolecules can be eluted by heating at least one of the magnetic beads or the nanothin film with the laser emitted from the light irradiator. As shown in the conceptual diagram in Figure 7, when the biomolecules are eluted from the magnetic beads, a high-concentration biomolecule field 120 is formed around the magnetic beads. Biomolecules present in the space where this high-concentration biomolecule field overlaps with the capture distance range pass through the nanopore.

[0057] (S5: Nanopore passage and measurement step) The eluted biomolecules are detected by the biomolecular detection function of this embodiment, i.e., by detecting changes in the electrical signal using the measurement unit. This can be measured by the eluted biomolecules moving by free diffusion from the first liquid tank to the second liquid tank, completely passing through the nanopore. Figure 8 shows an overview of the change in ionic current accompanying the movement of biomolecules in the liquid tank. The current value measured when the biomolecule passes through the nanopore (b) is smaller than when the biomolecule is present in the first liquid tank (a) and the second liquid tank (c). In this way, biomolecules can be detected using this embodiment. [Example]

[0058] Here, experiments were carried out on the biomolecule detecting device and biomolecule measuring method of the above embodiment to confirm their effects, which will be described in detail below.

[0059] (Biomolecular detection device) The flow cell was constructed from PEEK. The first (Trans) and second (Cis) liquid reservoirs contained 1M KCl and 25mM HEPES buffer solutions, respectively. The pH of the Trans reservoir was adjusted to 7, and the pH of the Cis reservoir was adjusted to 10. After nanopore formation, the pH of the Cis reservoir was adjusted to 7. A 50nm-thick SiN2 nanofilm was deposited by chemical vapor deposition on a 2µm SiO2 layer formed on a Si substrate, resulting in a Si / SiO2 / SiN2 laminated structure. Ag / AgCl electrodes were used as electrodes. A 532nm laser was used for the light irradiation section, along with a 40x objective lens (NA: 0.65), a Thorlabs, Inc., diaphragm shutter with a controller, and a Thorlabs, Inc., aluminum mirror. A Molecular Devices Axopatch 200b was used as the ammeter and power supply. The magnet was a Sonic magnetic clip, and was placed on the top surface of the flow channel cell, on the cis side. The flow channel cell was fixed to a microscope stage, which served as the flow channel cell installation stage. Green fluorescent protein (GFP) was used as the target molecule to be detected, and GFP-Trap (registered trademark) was used as the magnetic bead that could capture this.

[0060] (nanopore formation) First, we created a nanopore for molecular detection. A laser with a wavelength of 532 nm and an output of 91.9 mW was applied for 10 seconds. 7 W / cm 2A nanopore was formed by irradiating the SiN2 membrane with laser light and then applying a voltage of 1000 mV. The ionic current was measured using a Molecular Devices Axopatch 200b. The converted waveform was input into a PC, and the ionic current was read using a National Instruments LabVIEW program. Using this program, the sudden increase in current value that occurred upon nanopore formation was detected, and the voltage application and laser irradiation were stopped, forming a nanopore of several nanometers in diameter. After nanopore formation, a large instantaneous decrease in ionic current was confirmed by adding 1 μL of 2 kbp dsDNA at a concentration of 0.5 μg / μL to the cis side, confirming the formation of a nanopore capable of molecular detection. Hereafter, this large instantaneous decrease in ionic current is referred to as the "cutoff current."

[0061] (Magnetic bead movement near the nanopore) 2 μL of a suspension of GFP-conjugated magnetic beads was dropped onto the trans side, and a flow field was generated in the 1M KCl, 25 mM HEPES buffer solution on the trans side, dispersing the GFP-conjugated magnetic beads on the trans side. In this state, a magnet was brought close from the top of the cis side, and the magnetic beads were placed on the substrate surface. After placement, a focused laser with a wavelength of 532 nm and an output of 8.5 mW was applied for 10 min. 6 W / cm 2 The position of the magnetic beads was manipulated by bringing them close to the nanopore.

[0062] (GFP elution) After the magnetic beads were transferred, the laser output was changed to 45.3 mW, and a 10 6 W / cm 2 The laser is emitted at 1000mW, and then defocused to widen the laser irradiation range. 3 W / cm 2 Thermal dissolution was performed by laser heating.

[0063] (GFP-detected ion current) After elution of GFP, voltage was applied and the ionic current was measured. A large cutoff current of approximately 60-90% was observed for GFP-bound magnetic beads. The voltage was then changed from 150 to 300 mV in 50 mV increments, and applied for 5 minutes at each voltage. The frequency of the cutoff current increased with increasing applied voltage, indicating that the GFP eluted from the magnetic beads passed through the nanopore, which was detected as an ionic current. [Industrial Applicability]

[0064] The present invention can detect minute amounts of protein, DNA, and RNA, enabling genetic testing and protein detection in medical settings to be performed using extremely small amounts, such as a single drop of blood, from patients. It can also enable outdoor virus testing and ecosystem surveys to be performed using extremely small amounts. [Explanation of symbols]

[0065] 100: Low concentration detection device 101: Flow path cell 102:First electrolyte solution 103a, 103b: Electrode (AgCl electrode) 104: Nano-thin film 105: Nanopore 106: Magnetic beads 107: Biomolecules 108: Magnet 109: Flow path cell installation stage (microscope stage) 110: Computer 111:Ammeter 112: Power supply 113: Laser driver 114: Laser element 115: Shutter 116: Mirror 117: Objective lens 118: Focused laser 119: Unfocused laser 120: High concentration biomolecular field 121: Substrate (silicon) 122: Intermediate layer (silicon dioxide) 123: Cover glass 202: Second electrolyte solution

Claims

1. A biomolecule detection device including a flow path cell, a measurement unit, a light irradiation unit, and a magnet, the flow path cell includes a first liquid reservoir containing a first electrolyte solution, a second liquid reservoir containing a second electrolyte solution, and a nanomembrane having a nanopore separating the first liquid reservoir from the second liquid reservoir; magnetic beads having biomolecules bound thereto are provided in the first liquid vessel; the light irradiating unit includes a laser element that emits laser light of a wavelength that is absorbed by the nano-thin film and the magnetic beads to generate heat; the magnet is disposed outside the flow cell; The measurement unit has a function of detecting a change in an electrical signal that occurs when the biomolecules separated from the magnetic beads completely pass through the nanopore from the first liquid vessel and move into the second liquid vessel. A biomolecule detection device characterized by:

2. the light irradiation unit has a function of controlling the output density of the laser so that it is smaller when separating the biomolecules from the magnetic beads than when forming the nanopore in the nanomembrane, The laser irradiation position can be moved within a range where an electrical attractive force acts near the nanopore. The biomolecule detection device according to claim 1 .

3. the light irradiation unit has a feedback control function of controlling the output density of the laser in accordance with the measurement result of the measurement unit, The magnetic force from the magnet acts to bring the magnetic beads into contact with or close to the first liquid reservoir surface of the nanomembrane when separating the biomolecules from the magnetic beads, and does not act on the magnetic beads when forming the nanopore in the nanomembrane. The biomolecule detection device according to claim 1 .

4. A biomolecule detection device including a flow path cell, a measurement unit, a light irradiation unit, and a magnet, placing magnetic beads in contact with or adjacent to the surface of the first reservoir of the nano-thin film by the magnetic force of the magnet disposed outside the flow cell; placing the magnetic beads in an area where biomolecules are separated by a laser emitted from the light irradiation unit; a step of heating at least one of the magnetic beads and the nano-thin film that is in contact with or close to the magnetic beads with a laser emitted from the light irradiation unit to separate the biomolecules; moving the separated biomolecules from the first reservoir completely through the nanopore into a second reservoir; the range in which the biomolecules are separated is a range in which an electrical attractive force acts in the vicinity of the nanopore; The measurement unit detects a change in the electrical signal, thereby detecting the biomolecule. A biomolecule measurement method characterized by:

5. and forming nanopores by laser etching the nanomembrane placed in the flow path cell with a laser emitted from the light irradiation unit. The biomolecule measuring method according to claim 4 .

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