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WO2018142480A1 - Dispositif d'analyse biomoléculaire et élément de fixation de biomolécule - Google Patents

Dispositif d'analyse biomoléculaire et élément de fixation de biomolécule Download PDF

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Publication number
WO2018142480A1
WO2018142480A1 PCT/JP2017/003456 JP2017003456W WO2018142480A1 WO 2018142480 A1 WO2018142480 A1 WO 2018142480A1 JP 2017003456 W JP2017003456 W JP 2017003456W WO 2018142480 A1 WO2018142480 A1 WO 2018142480A1
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WIPO (PCT)
Prior art keywords
substrate
biomolecule
nanopore
fixing member
individual
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PCT/JP2017/003456
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English (en)
Japanese (ja)
Inventor
玲奈 赤堀
武田　健一
善光 柳川
佑介 後藤
一真 松井
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Hitachi High Tech Corp
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Hitachi High Technologies Corp
Hitachi High Tech Corp
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Priority to PCT/JP2017/003456 priority Critical patent/WO2018142480A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to a biomolecule analysis device and a biomolecule fixing member.
  • next-generation DNA sequencers a technique for electrically directly measuring the base sequence of a biomolecule (biological polymer) such as a DNA chain without performing an extension reaction or a fluorescent label has attracted attention.
  • a biomolecule biological polymer
  • This method is a method in which a DNA sequence is directly measured and a base sequence is determined without using a reagent.
  • individual base species contained in a DNA strand passing through nanometer-order pores (hereinafter referred to as “nanopores”) formed in a thin film are directly measured by the amount of blocking current, and the base species are sequentially identified.
  • the template DNA is not amplified by the enzyme, and a label such as a phosphor is not used. For this reason, it is expected as a method capable of high-throughput, low running cost, and long base DNA decoding.
  • One of the problems of the nanopore method is the control of DNA transport through the nanopore.
  • the DNA nanopore passage time is set to 100 ⁇ s or more per base from the current noise at the time of measurement and the time constant of DNA molecule fluctuation. There are thought to be.
  • DNA nanopore passage time is usually as short as 1 ⁇ s or less per base, and it is difficult to sufficiently measure the blocking current derived from each base.
  • DNA ends are fixed to a parallel flat plate Si probe (fixed substrate) larger than the thin film of the nanopore substrate, and the micro displacement in the z direction of the fixed substrate is controlled by an external drive mechanism.
  • an external drive mechanism There is one that introduces DNA into the nanopore without positioning the nanopore and arbitrarily controls the passing speed of the DNA passing through the nanopore. Since DNA is negatively charged in an aqueous solution, it receives a force due to a potential difference generated in the vicinity of the nanopore and is introduced into the nanopore. At the same time, by monitoring the ion current passing through the nanopore, it is possible to obtain a blocking current signal when DNA passes through the nanopore.
  • dsDNA double-stranded DNA
  • ssDNA single-stranded DNA
  • ssDNA single-stranded DNA
  • a certain degree of tilt may occur on the opposing surfaces of the nanopore substrate and the fixed substrate.
  • a DNA introduction possible region (about 300 nm) generated in the vicinity of the nanopore. If the size of the nanopore substrate increases due to the array of nanopores, the distance between the nanopore at the end of the nanopore substrate and the fixed substrate becomes 300 nm or more due to the inclination with the fixed substrate, and there is a region where DNA cannot be introduced. End up. This results in a decrease in measurement throughput. Even if biomolecules can be introduced into the nanopore while the bias is generated, the measurement environment is biased due to the nonuniformity of the substrate approach distance, which causes an error in the measurement signal.
  • the biomolecule analysis device of the present invention is, for example, a nanopore substrate having a thin film on which a plurality of nanopores are formed, a common tank provided on the top of the nanopore substrate, and a lower portion of the nanopore substrate, A plurality of individual tanks communicated with the common tank through the nanopore, a common electrode disposed in the common tank, an electrode substrate on which a plurality of individual electrodes respectively disposed in the plurality of individual tanks are formed, and a living body
  • a biomolecule fixing member that fixes molecules and is driven toward the nanopore substrate in the common tank, and a tilt correction mechanism that is disposed below the biomolecule fixing member or the electrode substrate. It is deformed by the applied pressure generated when the biomolecule fixing member is pressed against the nanopore substrate, and the deformed shape is maintained.
  • the biomolecule fixing member is driven toward the nanopore substrate, and the tilt correction mechanism is deformed by the applied pressure generated by pressing the biomolecule fixing member against the nanopore substrate. I do.
  • the biomolecule fixing member of the present invention is disposed between a biomolecule fixing substrate having a biomolecule fixing surface, a drive mechanism connecting member for connecting to the driving mechanism, and the biomolecule fixing substrate and the driving mechanism connecting member.
  • the tilt correction mechanism is deformed by the pressure applied between the biomolecule fixing substrate and the drive mechanism connecting member, and maintains the deformed shape.
  • the biomolecule analysis device includes, as one aspect, a nanopore substrate having a thin film on which a plurality of nanopores are formed, one common tank provided on the top of the nanopore substrate, and a lower portion of the nanopore substrate.
  • Sensors are provided in at least three locations on the peripheral edge of the nanopore substrate, and the sensors are provided in a region where the biomolecule fixing member approaches and a thin film having no nanopores is exposed.
  • the phase change amount of the difference between the current that flows when an AC voltage is applied between and the current that flows when an AC voltage is applied between the individual electrode and the common electrode of the second individual tank is monitored.
  • the biomolecule analysis device includes, as one aspect, a nanopore substrate having a thin film on which a plurality of nanopores are formed, one common tank provided on the top of the nanopore substrate, and a lower portion of the nanopore substrate.
  • a plurality of individual tanks each communicating with the common tank through nanopores, a common electrode disposed in the common tank, and a plurality of individual electrodes respectively disposed in the plurality of individual tanks
  • the tilt correction mechanism disposed below the electrode substrate, and the biomolecule fixing member that is driven toward the nanopore substrate by fixing the biomolecules, the peripheral edge of the nanopore substrate And the tilt correction mechanism is deformed by the applied pressure generated when the biomolecule fixing member is pressed against the nanopore substrate, and the shape after the deformation is obtained. It is intended to maintain.
  • the biomolecule analysis device includes, as one aspect, a nanopore substrate having a thin film on which a plurality of nanopores are formed, one common tank provided on the top of the nanopore substrate, and a lower portion of the nanopore substrate.
  • a nanopore substrate having a thin film on which a plurality of nanopores are formed, one common tank provided on the top of the nanopore substrate, and a lower portion of the nanopore substrate.
  • a device for analyzing biomolecules which is used for analyzing biomolecules fixed to a biomolecule fixing member that has a correction mechanism and is driven toward a nanopore substrate in a common tank, for detecting the approach of the biomolecule fixing member
  • Sensors are provided at at least three locations on the periphery of the nanopore substrate, and the sensors are arranged on the periphery of the nanopore substrate and the nanopore provided in the region where the biomolecule fixing member approaches.
  • the through hole of large dimensions Ri is to monitor the changes in the ionic current flowing through the through hole.
  • the inclination of the biomolecule fixing member generated when the biomolecule fixing member approaches the nanopore substrate is eliminated, so that the biomolecule fixed to the biomolecule fixing member becomes the nanopore of the nanopore substrate.
  • the probability of being introduced is made uniform in the plane.
  • the cross-sectional schematic diagram which shows an example of the biomolecule analyzer which provided the inclination correction mechanism in the fixing member.
  • the schematic sectional drawing which showed a part of process of providing a gold stud bump in a fixing member.
  • the top view and sectional drawing which show the example of a shape of the bump immediately after bonding gold
  • the top view and sectional drawing which show the example of a shape of the bump after pressing a Si substrate.
  • FIG. 6 is a schematic cross-sectional view showing a device configuration of a conventional array-type biomolecule analyzer.
  • FIG. 6 is a schematic cross-sectional view showing the configuration of a conventional array-type biomolecule analyzer.
  • an inclination correction mechanism is formed in a biomolecule fixing member (hereinafter also simply referred to as a fixing member) provided with a biomolecule fixing substrate (hereinafter also simply referred to as a fixing substrate). It was found that the relative inclination between the nanopore substrate and the fixed substrate can be eliminated by contacting and pressing the fixing member with the nanopore substrate of the biomolecule analysis device for analyzing the characteristics of the biomolecule.
  • the tilt correction mechanism provided on the fixed member satisfies the following conditions (a) to (d).
  • (A) Correctable immediately before measurement (b) Constructed by a plastically deformable material having malleable ductility (c) Adhesive force is 1 gf or more (d) Rigidity, force of 0.1 N or less In such a case, deformation does not occur.
  • Adhesive force is 1 gf or more
  • Rigidity, force of 0.1 N or less In such a case, deformation does not occur.
  • in contact with the nanopore substrate in order to detect that the relative tilt between the nanopore substrate and the fixed substrate has been eliminated, at the positions corresponding to the four corners of the fixed substrate on the nanopore substrate, It is preferable to provide an approach detection mechanism.
  • a device for biomolecule analysis used when analyzing a biomolecule composed of nucleic acids separates the first and second liquid tanks filled with the electrolyte solution from the first and second liquid tanks, respectively.
  • the device for biomolecule analysis can be configured as an array device.
  • An array device refers to a device having a plurality of sets of liquid chambers partitioned by a nanopore substrate.
  • the first liquid tank is a common tank
  • the second liquid tank is a plurality of individual tanks.
  • an electrode is arranged in each of the common tank and the individual tank.
  • the biomolecule analyzer has a measuring unit that measures an ionic current (blocking signal) flowing between electrodes provided in the biomolecule analyzing device, and is based on the value of the measured ionic current (blocking signal). This is a device for acquiring sequence information of biomolecules.
  • Biomolecules are fixed to a fixed substrate of a fixing member, and are transported and approached by a driving mechanism directly above the nanopore substrate through an opening provided in the upper part of the common tank of the biomolecule analysis device.
  • a driving mechanism directly above the nanopore substrate through an opening provided in the upper part of the common tank of the biomolecule analysis device.
  • the parallelism between the fixed substrate and the nanopore substrate is maintained, and the measurement yield when using a plurality of individual tanks at once in the array device is improved.
  • the above-mentioned fixing member when the fixed substrate is brought into contact with the nanopore substrate of the biomolecule analysis device, the two who have non-uniform contact points when the correction mechanism is not used are It was confirmed that proper contact was achieved.
  • the fixed substrate constitutes a fixed member together with the drive mechanism connecting member and the tilt correcting mechanism.
  • a fixing member with a tilt correction mechanism By using a fixing member with a tilt correction mechanism, the relative tilt between the nanopore substrate in the biomolecule analysis device and the fixed substrate in the fixing member is eliminated, and the nanopore substrate in the biomolecule analysis device is uniformly distributed to all nanopores. Biomolecules can be introduced to improve the measurement yield. Below, it demonstrates concretely with reference to drawings.
  • FIG. 18 is a schematic cross-sectional view showing the configuration of a conventional array-type biomolecule analyzer.
  • the biomolecule analysis device 100 includes an electrode substrate 113 on which a plurality of individual electrodes 115B are arranged, a nanopore substrate 111 on which a plurality of nanopores 110 are formed, and an outer wall 114 for forming a common tank 104 on top of the nanopore substrate 111.
  • an electrode substrate 113 on which a plurality of individual electrodes 115B are arranged
  • a nanopore substrate 111 on which a plurality of nanopores 110 are formed
  • an outer wall 114 for forming a common tank 104 on top of the nanopore substrate 111.
  • a plurality of individual tanks 105 are provided between the nanopore substrate 111 and the electrode substrate 113.
  • One common electrode 115 ⁇ / b> A is disposed in the common tank 104, and one individual electrode 115 ⁇ / b> B is disposed in each of the plurality of individual tanks 105.
  • the common tank and the individual tanks are filled with the electrolyte solution.
  • a voltage is applied from the power source 120 between the common electrode 115A and the individual electrode 115B, and an ionic current flows through the nanopore 110.
  • no biomolecule is introduced into the nanopore 110, so that an electric current corresponding to the size (pore diameter) of the nanopore is measured by the ammeter 121.
  • the measurement value of the ammeter 121 is output to the computer 130.
  • the end-modified DNA strand 116 is fixed to the fixing member 117, and the fixing member 117 is brought close to the nanopore substrate 111 by a driving mechanism. Then, the DNA strand 116 fixed to the fixing member 117 detects a potential gradient formed in the vicinity of the nanopore and is introduced into the nanopore 110.
  • the fixing member 117 when the fixing member 117 is brought close to the nanopore substrate 111, there is a case where an inclination exists between the fixing member and the nanopore substrate, so that the opposing surfaces may not be parallel as shown in FIG.
  • the parallel projection to the fixing surface of the driving mechanism is insufficient.
  • the surface of the nanopore substrate 111 is inclined when the biomolecule analysis device 100 is installed because the electrode substrate 113 itself of the biomolecule analysis device 100 has an inclination.
  • the distance at which DNA is introduced by the potential gradient generated around the nanopore of the nanopore substrate is about 300 nm from the center of the nanopore.
  • the fixing member is separated from the nanopore substrate by a maximum of 2.4 ⁇ m when tilted by 0.1 degree. Accordingly, there is a possibility that DNA on the fixing member is not introduced into some nanopores of the nanopore substrate.
  • the probability of introducing DNA into a plurality of nanopores provided on the nanopore substrate needs to be 90% or more.
  • FIG. 1 is a schematic cross-sectional view showing an embodiment of a biomolecule analyzer in which an inclination correction mechanism is provided on a fixed member.
  • FIG. 2 is a diagram for explaining an example of an arrangement place of the plastically deformable material, and is a schematic top view and a schematic cross-sectional view showing an enlarged portion of the fixing member and the nanopore substrate.
  • a plastic deformation material is provided inside the biomolecule fixing member 117. That is, the fixing member 117 of this embodiment has a biomolecule fixing substrate 117A that has a biomolecule fixing surface and can fix a biomolecule, and a driving mechanism connecting member 117B for connecting to the driving mechanism. It has a plastic deformation material as the inclination correction mechanism 117C.
  • the fixing member 117 having such a structure the plastic deformation material is deformed by using a force pressing the fixing member 117 against the nanopore substrate 111. Thereby, the inclination between the fixed substrate 117A and the nanopore substrate 111 can be eliminated, and the surface of the fixed substrate 117A can be made parallel to the surface of the nanopore substrate 111.
  • the pressing force against the plastically deformable material a force generated by contact with the nanopore substrate 111 when the fixing member 117 is lowered using the driving mechanism is used.
  • the nanopore substrate 111 has a structure in which a thin film 111A in which the nanopore 110 is opened is sandwiched between a lower thin film fixing member 111C and an upper thin film fixing member 111B.
  • an upper thin film fixing member 111B is formed on the thin film 111A, and an opening is formed at the position of the nanopore by lithographic processing to minimize the area where the thin film is exposed.
  • a dotted line frame 310 in FIG. 2 represents the outline of the opening processed in the upper thin film fixing member 111B.
  • a dotted line frame 311 is a position where an etch stop position obtained by taper etching the lower thin film fixing member 111C made of a silicon substrate intersects the thin film 111A.
  • the thin film 111A is not damaged by the amount of pressing on the upper thin film fixing member 111B outside the dotted line frame 311. That is, the formation position of the plastically deformable material is a position where the force at the time of contact is well transmitted, that is, an area where the thin film is not exposed, and an area on the upper thin film fixing member 111B.
  • the material used for the inclination correction mechanism 117C needs to be a material suitable for fine conveyance control, a certain degree of rigidity is required. Therefore, in this embodiment, as the inclination correction mechanism 117C, the plastic deformation that can be deformed by the applied pressure generated when the fixed substrate 117A is brought into close contact with the nanopore substrate 111 by the driving mechanism and can maintain the shape after the deformation. Material was used.
  • a material that maintains a deformed shape by pressurization and that can maintain stable displacement in active measurement is a gold stud bump.
  • the principle was verified by adopting an array of stud bumps made of gold as the tilt correction mechanism 117C.
  • the effect of eliminating the tilt can be obtained by deforming the stud bump made of gold by the pressure generated when the fixing member and the nanopore substrate are brought into close contact with each other by the drive mechanism.
  • FIG. 3 is a schematic cross-sectional view showing a part of the process of providing the gold stud bump in the fixing member. The experiment was conducted with the dimensions of the fixed substrate being 6 mm ⁇ 12 mm.
  • FIG. 4 is a top view and a cross-sectional view showing an example of the shape of the stud bump 414A immediately after gold is bonded.
  • FIG. 5 is a top view and a cross-sectional view showing an example of the shape of the stud bump 414B after the Si substrate is pressed. 4 and 5 show a state in which a bump having a height of 72 ⁇ m is deformed to a height of 30 ⁇ m by pressing and a flat surface of 30 ⁇ m can be formed.
  • a 20 mm square fixed substrate is used according to the array device dimensions, and the required contact pressure is 670 gf.
  • the required pressing strength increases as the number of bumps increases.
  • the required number of bumps to be formed when using a fixed substrate for an array device is not less than 600 gf, which is the holdable strength, and not more than the pushable strength. Since this condition varies depending on the bump size and gold density, if the above deformation and holding are possible, it should be designed according to the necessary conditions.
  • FIG. 6 and 7 are diagrams showing the results.
  • FIG. 6 is a diagram showing a contact area when a fixing member without an inclination correction mechanism is used
  • FIG. 7 is a diagram showing a contact area when a fixing member with a bump is used as the inclination correction mechanism.
  • the broken line indicates the pressing position of the fixed substrate on which the bumps are installed.
  • the pressing strength is about 1N, which is the combined weight of the piezo holder and the piezo.
  • the size of the fixed substrate for array devices may be 15 to 20 mm, and the tilt of the fixed substrate and the nanopore substrate may occur at a maximum of about 0.1 °. In this case, the tilt cancellation distance is 35 ⁇ m. It becomes.
  • the dimensions that can be set by the bumps are 70 to 90 ⁇ m, and the height achieved when crushing with 10 N is about 5 ⁇ m. Therefore, the height that can be eliminated at this time is 65 to 85 ⁇ m. In this case, it is necessary to eliminate the inclination of 35 ⁇ m at the maximum, but it can be seen that it is in a sufficiently resolvable range.
  • the pressure generated during crushing should be kept to the extent that the nanopore substrate is not damaged. Since the plastic deformation material used in this embodiment as the tilt correction mechanism is provided on the fixed substrate, the rigidity of the tilt canceling material can affect the transport controllability of biomolecules. Therefore, a high rigidity is required as a non-pressurized state. On the other hand, it is necessary to be able to be plastically deformed within the above range, so it is preferable to use a metal having malleability and ductility. Gold, silver, platinum, copper, lead, tin, nickel, or alloys thereof are suitable as specific materials for realizing such plastic deformation.
  • a means for confirming that the inclination between the fixed substrate and the nanopore substrate has been eliminated will be described. Since excessive pressurization causes damage to the nanopore substrate, a sensor that detects the approach of the fixing member to the nanopore substrate is used as a means of monitoring that the tilt has been eliminated in order to keep the pressurization to a level that does not cause damage. It is preferable to provide it.
  • FIG. 8 is a diagram showing the relationship between the relative distance between the nanopore substrate and the fixed substrate and the ionic current flowing through the large-diameter pore.
  • (B) in the figure shows the relationship at a position where the fixing member is not completely in contact with the nanopore substrate. Thereafter, when the fixing member is brought close to the nanopore substrate, the space between the nanopore substrate and the fixing member is narrowed, so that the space formed by the nanopore substrate and the fixing member becomes a resistance, resulting in a decrease in output current.
  • the acquired current value approaches a constant value.
  • FIG. 9 and 10 are schematic diagrams showing the cause of the generation of the ionic current value shown in FIG.
  • FIG. 9 shows the cause of generation of the ionic current value measured at time (b) before the fixing member 117 approaches the nanopore substrate 111.
  • Nanopore substrate - the distance d between the fixing substrate is d b.
  • the current value I b where the through-hole and a resistor 301 between the individual electrode 115B for proximity detection and the common electrode 115A is measured.
  • the resistor 301 resulting from the through hole, the fixing member 117 is close to the nanopore substrate 111 As a result, the resistance component 302 generated by the narrow conductive path between the fixing member 117 and the nanopore substrate 111 is overlapped, so that the measured ion current Ia is reduced.
  • through holes large-diameter pores
  • the approach of the fixing member 117 to the nanopore substrate 111 is detected from the change in the ionic current value flowing therethrough.
  • a specific configuration example for realizing proximity detection by forming a large-diameter pore on the nanopore substrate as described above is shown below.
  • a SiO / SiN film was further formed with a film thickness of 250 nm / 100 nm on the Si substrate.
  • a SiN film of 100 nm was formed on the back surface, and an opening of 1 mm ⁇ was formed.
  • TMAH TMAH
  • a free-standing film of SiO / SiN is formed with 65 ⁇ m square. Thereafter, by removing the free-standing film, a through hole of 65 ⁇ m ⁇ can be formed in the Si substrate.
  • the liquid baths above and below the Si substrate were filled with KCl solution, and an AgCl electrode was provided in each liquid bath to monitor the current value. The measured current value was 4 ⁇ A when 0.1 V was applied.
  • the mechanism for measuring the ionic current flowing through the large-diameter pore for the purpose of approach detection is basically the same as the ammeter for measuring the characteristics of biomolecules, so it can be prepared in parallel.
  • the current value measured with the large-diameter pore is about three orders of magnitude larger than the current value measured with the nanopore, it is preferable to reduce the amplification factor of the ammeter by three orders of magnitude.
  • the device for biomolecule analysis has a nanopore substrate.
  • FIG. 11 and 12 are schematic cross-sectional views showing a configuration example of the biomolecule analyzer.
  • the figure shows a configuration example of a biomolecule analyzer having a disposable biomolecule analysis device 100, power supplies 120 and 123, ammeters 121 and 124, and a computer 130.
  • the biomolecule analyzer of the present embodiment has an inclination correction mechanism and an approach detection mechanism.
  • FIG. 11 shows a state before the inclination correction
  • FIG. 12 shows a state where the inclination correction is completed.
  • the biomolecule analysis device 100 includes upper and lower liquid tanks separated by a nanopore substrate 111.
  • the nanopore substrate 111 includes a thin film 111A on which the nanopore 110 is formed, and an upper thin film fixing member 111B and a lower thin film fixing member 111C formed so as to sandwich the thin film.
  • the nanopore 110 may be formed at any position of the thin film 111A.
  • the upper liquid tank is a common tank 104, and the lower liquid tank is further divided into a plurality of liquid tanks.
  • the lower thin film fixing member 111 ⁇ / b> C has four spaces separated by three partition walls, and these spaces are respectively used as the individual tanks 105.
  • the common tank 104 is used as a common liquid tank for the four individual tanks 105 located on the lower side. Each individual tank 105 communicates with the common tank 104 via the nanopore 110.
  • the upper thin film fixing member 111B and the thin film 111A constitute a part of the structure of the common tank 104. Further, the thin film 111 ⁇ / b> A and the lower thin film fixing member 111 ⁇ / b> C constitute a part of the structure of the individual tank 105. Both the common tank 104 and the individual tank 105 are filled with the electrolyte solution.
  • the volume of the electrolyte solution is on the order of microliters or milliliters.
  • KCl, NaCl, LiCl, CsCl, or MgCl 2 is used for the electrolyte solution.
  • the dimension of the thin film 111A exposed at the opening provided in the upper thin film fixing member 111B and the lower thin film fixing member 111C is an area where two or more nanopores are difficult to be formed when nanopores are opened by voltage application, and The area needs to be acceptable in terms of strength.
  • the area is, for example, about 100 to 500 nm ⁇ .
  • the film thickness is suitably about 7 nm or less that can form the nanopore 110 having an effective film thickness equivalent to one base.
  • Each individual tank 105 is provided with a single nanopore 110 and an individual electrode 115B, which are insulated from each other by a partition wall.
  • a common electrode 115 ⁇ / b> A is disposed in the common tank 104. For this reason, the electric current which flows through each nanopore 110 can be measured independently.
  • the common electrode 115A and the individual electrodes 115B are, for example, Ag, AgCl, and platinum, and are in contact with the electrolyte solution.
  • a connection terminal electrically connected to the common electrode 115 ⁇ / b> A and the individual electrode 115 ⁇ / b> B is provided on the outer peripheral surface of the biomolecule analysis device, and is connected to the power source 120 and the ammeter 121.
  • the ammeter 121 includes an amplifier that amplifies a current flowing between the electrodes by applying a voltage and an ADC (Analog-to-Digital Converter). A detection value that is an output of the ADC is output to the computer 130. The computer 130 collects and records the detected current value. As shown in FIG. 11, the power source 120, the ammeter 121, and the computer 130 are not separate members from the biomolecule analysis device 100, but the power source 120, the ammeter 121, and the computer 130 are the biomolecule analysis device. 100 may be integrated.
  • An opening 501 is formed in a part of the outer wall 114 in the common tank 104 constituting the biomolecule analysis device 100, and the drive mechanism 201 is attached to the opening 501.
  • a fixing member 117 is attached to the lower surface of the drive mechanism 201 by a drive mechanism connecting member 117B.
  • the fixing member 117 has a drive mechanism connecting member 117B, a fixed substrate 117A, and an array of gold stud bumps as an inclination correction mechanism 117C provided therebetween.
  • a DNA chain 116 is fixed to the surface of the fixing substrate 117A facing the nanopore substrate 111 of the fixing member 117.
  • the size of the fixed substrate 117A on which the DNA strand 116 is fixed is larger than the size of the portion of the thin film 111A that is in contact with the electrolyte solution.
  • the fixing member 117 is driven in the vertical direction in the figure by the drive mechanism 201 toward the nanopore substrate 111 in the common tank 104. That is, the surface of the fixed substrate 117A to which the DNA strand 116 is fixed is driven in a direction toward or away from the nanopore substrate 111.
  • the DNA strand 116 is introduced into the nanopore 110 when the surface of the fixed substrate 117A approaches the nanopore substrate 111.
  • the operation of the drive mechanism 201 is controlled by the control unit 202.
  • Contact between the fixed substrate 117A and the thin film 111A is prevented by the upper thin film fixing member 111B. This is because if the fixed substrate 117A comes into contact with the thin film 111A on which the nanopores 110 are formed, the thin film 111A may be destroyed. That is, the upper thin film fixing member 111B also functions as means for stopping the lowering of the fixed substrate 117A. After the upper thin film fixing member 111B is formed on the upper part of the thin film 111A, an opening is formed by patterning and etching a part thereof.
  • the nanopore 110 is formed by dielectric breakdown at the opening of the upper thin film fixing member 111B, that is, at the place where the thin film is formed in the nanopore substrate 111.
  • the combined film thickness of the upper thin film fixing member 111B and the lower thin film fixing member 111C is 200 to 200 considering the securing of the strength of the thin film 111A and the fixed height fluctuation of the biomolecule fixed on the surface of the fixed substrate 117A.
  • About 500 nm is appropriate.
  • the thin film 111A has a diameter of 500 nm
  • the upper thin film fixing member 111B has a film thickness of 250 nm
  • the lower thin film fixing member 111C has a film thickness of 100 nm.
  • the fixing member 117 can be fixed to the drive mechanism 201 by vacuum suction or pressure bonding.
  • the drive mechanism 201 is made of a piezoelectric material typified by a piezo element, and can be driven at 0.1 nm / s or more.
  • As the piezoelectric material barium titanate (BaTiO 3 ), lead zirconate titanate (PZT), zinc oxide (ZnO), or the like is used.
  • the end of the DNA strand 116 and the surface of the fixed substrate 117A are bonded to each other by a covalent bond, an ionic bond, an electrostatic interaction, a magnetic force, or the like.
  • a DNA strand is immobilized by a covalent bond
  • a DNA strand whose end is modified via APTES or glutaraldehyde is used.
  • Si and SiO serving as a scaffold for APTES are formed on the surface of the fixed substrate 117A in order to use the above bond.
  • the fixing member 117 When the surface of the nanopore substrate 111 and the surface of the fixed substrate 117A are not parallel to each other but tilted, if the fixing member 117 is lowered toward the nanopore substrate 111 by the drive mechanism 201, the fixed substrate 117A is first shown as shown in FIG. Is in contact with the upper thin film fixing member 111B of the nanopore substrate 111. When the fixing member 117 is further lowered in this state and the fixing member 117 is pressed against the nanopore substrate 111, the tilt correction mechanism 117C provided inside the fixing member 117 is deformed by the pressure generated or the pressure.
  • the amount of deformation of the tilt correction mechanism 117C varies depending on the location, and by deforming the tilt correction mechanism 117C, the surface of the nanopore substrate 111 and the surface of the fixed substrate 117A are finally made parallel to each other as shown in FIG. Once the tilt correction mechanism 117C is deformed by receiving pressure, it maintains its deformed shape.
  • through holes 502 having the same opening diameter as the opening of the upper thin film fixing member 111B are formed in at least three, preferably four corners, of the region where the fixing substrate 117A approaches at the periphery. .
  • the ion current flowing through the through-hole 502 having a size larger than that of the nanopore is monitored by the ammeter 124 to detect the approach between the fixed substrate 117A and the nanopore substrate 111. That is, the individual electrode 115 ⁇ / b> B arranged in the individual tank 105 having the through hole 502 is used as a sensor for detecting the approach of the fixed substrate 117 ⁇ / b> A to the nanopore substrate 111.
  • the voltage applied between the common electrode 115A and the individual electrode 115B from the power source 123 is adjusted according to the detection system.
  • the principle of approach detection is as described with reference to FIGS. For example, when the current values detected by the proximity sensors provided at the above four corners decrease and become equal values, it is determined that the surface of the fixed substrate 117A is parallel to the surface of the nanopore substrate 111, The driving of the fixing member 117 by the driving mechanism 201 is stopped.
  • the DNA strand 116 is introduced into the plurality of nanopores at a desired speed, and the blocking current is measured by the plurality of individual electrodes 115B, respectively. Perform analysis in parallel.
  • the inclination correction mechanism 117C is deformed as shown in FIG. 12, it has an adhesive force of 1 gf or more, has rigidity, and is not deformed by a force of 0.1 N or less.
  • the fixed substrate 117A can move integrally with the drive mechanism 201, and high-precision analysis is possible.
  • the fixing member 117 and the driving mechanism 201 are attached to the biomolecule analysis device 100. However, these members may not be attached at the distribution stage.
  • the thin film 111A on which the nanopore 110 is formed may be a lipid bilayer (biopore) composed of an amphipathic molecular layer in which a protein having a pore at the center is embedded, or from a material that can be formed by a semiconductor microfabrication technique. It may be a thin film (solid pore). Examples of materials that can be formed by semiconductor microfabrication technology include silicon nitride (SiN), silicon oxide (SiO 2 ), silicon oxynitride (SiON), hafnium oxide (HfO 2 ), molybdenum disulfide (MoS 2 ), and graphene. is there.
  • the thickness of the thin film is 1 to 200 nm, preferably 1 to 100 nm, more preferably 1 to 50 nm, for example, about 5 nm.
  • the size of the nanopore can be appropriately selected according to the type of biological polymer to be analyzed, and is, for example, 0.9 nm to 100 nm, preferably 0.9 nm to 50 nm, specifically about 0.9 nm or more. , 10 nm or less.
  • the diameter of the nanopore used for analysis of ssDNA having a diameter of about 1.4 nm is preferably about 1.4 nm to 10 nm, more preferably about 1.4 nm to 2.5 nm, specifically about 1.6 nm. is there.
  • the diameter of the nanopore used for analyzing dsDNA having a diameter of about 2.6 nm is preferably about 3 nm to 10 nm, more preferably about 3 nm to 5 nm.
  • the depth of the nanopore can be adjusted by adjusting the thickness of the thin film 111A.
  • the depth of the nanopore is preferably less than the size of a single base in order to decompose the characteristics of biomolecules.
  • a thickness up to about 5 times the monomer unit constituting the living body polymer is allowed.
  • the depth of the nanopore is preferably 3 or more bases, for example, about 1 nm or more.
  • the interval at which the plurality of nanopores are arranged can be 0.1 ⁇ m to 10 ⁇ m, preferably 0.5 ⁇ m to 4 ⁇ m, depending on the electrode used and the ability of the electrical measurement system.
  • the method of forming the nanopore 110 in the thin film 111A is not particularly limited, and for example, electron beam irradiation by a transmission electron microscope or the like, or dielectric breakdown by voltage application can be used. For example, the method described in “Itaru Yanagi et al., Sci. Rep. 4, 5000 (2014)” can be used. In addition to applying a pulse voltage, nanopores can also be formed by electron beam irradiation with TEM (A. J. Storm et al., Nat. Mat. 2 (2003)).
  • the common tank and the individual tank that can store the measurement solution in contact with the thin film 111A can be appropriately provided with materials, shapes, and sizes that do not affect the measurement of the blocking current.
  • the measurement solution is injected so as to come into contact with the thin film 111A partitioning these liquid tanks.
  • the common electrode 115A and the individual electrode 115B are preferably made of a material capable of performing an electron transfer reaction (Faraday reaction) with an electrolyte in a measurement solution.
  • the silver halide or the alkali silver halide is used. It is made with. From the viewpoint of potential stability and reliability, it is preferable to use silver or silver-silver chloride.
  • FIG. 13 is a flowchart illustrating an example of a procedure from the tilt correction performed by the biomolecule analyzer to the measurement.
  • Step S11 First, a fixing member sandwiching plastic deformation materials as an inclination correction mechanism is prepared.
  • Step S12 A biomolecule is fixed to the fixing substrate of the fixing member.
  • Step S13 The fixing member is connected to the drive mechanism via a connection member in the fixing member.
  • Step S14 Thereafter, the driving mechanism is driven under the control of the control unit to bring the fixing member closer to the nanopore substrate. Alternatively, the tilt is eliminated by bringing the fixing member into contact with the nanopore substrate and pressing it.
  • Step S15 The proximity sensor is used to detect the proximity using the through-hole (large-diameter pore) of the nanopore substrate in the example of FIG. For example, when the conductance of the through holes provided at the four corners reaches the lower limit and no change is observed in the value of the ionic current flowing through the four through holes, the pressing of the fixing member is canceled, and the process proceeds to step S16. At this time, it is determined that the posture of the fixed substrate with respect to the nanopore substrate has changed from the state of FIG. 11 to the state of FIG. That is, it is determined that the biomolecule fixing surface of the fixing member is substantially parallel to the surface of the nanopore substrate.
  • Step S16 Biomolecules fixed on the fixed substrate are introduced into the nanopore by applying a voltage between the common electrode and the individual electrodes, and the biomolecules are analyzed by measuring the blocking current by moving the drive mechanism up and down. . Tilt detection and correction between the nanopore substrate and the fixed substrate are performed before biomolecule analysis as described above, but can also be performed during biomolecule analysis, and the fixed substrate is closest to the nanopore substrate. In this case, it is possible to correct the inclination again.
  • the fixing member and the biomolecule analysis device for analyzing the biological polymer according to the present embodiment include the above-described configuration as an element.
  • the fixing member and the biomolecule analysis vice may be provided together with instructions describing the use procedure, the use amount, and the like.
  • the fixing member may be provided in a state where it can be used immediately, or may be provided in a state where only the biomolecule to be measured is not bound. Such forms and preparation can be understood by those skilled in the art.
  • the device for biomolecule analysis may be provided in a state in which nanopores are formed in a state where it can be used immediately, or may be provided in a state in which it is formed at a provision destination.
  • FIG. 14 is a schematic cross-sectional view showing a configuration example of a biomolecule analyzer in which an inclination correction mechanism is arranged below the biomolecule analysis device.
  • a hard flat plate member 401 such as a silicon substrate is installed below the electrode substrate 113 of the biomolecular analysis device, and a gold stud bump is used as the tilt correction mechanism 402 between the flat plate member 401 and the electrode substrate 113.
  • the relative inclination of the biomolecule fixing member 117 and the nanopore substrate 111 is adjusted using the flat plate member 401 as a reference.
  • the fixing member 117 in the common tank 104 is lowered toward the nanopore substrate 111 by the driving mechanism, the lower right corner of the fixing member 117 first contacts the nanopore substrate 111 and the right side of the nanopore substrate 111 is moved to the right side. Press down.
  • the pressure applied by the pressing acts on the tilt correction mechanism 402 via the electrode substrate 113, and the bumps are deformed to reduce the height.
  • the tilt correction mechanism 402 is greatly deformed, and the left bump is also deformed sequentially, and the electrode substrate 113 is tilted.
  • the tilt correction mechanism 402 is deformed until the surface of the nanopore substrate 111 is parallel to the surface of the fixing member 117, and the electrode substrate 113 and the biomolecule analysis device 100 as a whole are tilted.
  • the timing for ending the pressing of the fixing member 117 by the drive mechanism is determined from the monitoring result of monitoring the change in the ionic current flowing through the through hole 502.
  • the bump array When the bump array is arranged on the lower surface of the biomolecule analysis device 100, it is not necessary to examine the strength of the adhesive force of the inclination correction mechanism 402.
  • the dimensions of the electrode substrate 113 vary depending on the required circuit quantity, but are at least larger than the nanopore substrate 111.
  • the assumed size of the nanopore substrate 111 is 30 cm ⁇ 30 cm, and therefore the inclination likelihood to be adjusted can be about 100 ⁇ m. Therefore, the height of the bump constituting the tilt correction mechanism 402 is required to be about 100 ⁇ m.
  • Example 3 In addition to monitoring the ionic current flowing through the through-holes provided in the nanopore substrate, a method of applying an AC bias is also possible as a proximity sensor for detecting tilt cancellation between the nanopore substrate and the fixed substrate of the biomolecule analysis device. . In this embodiment, the amount of change in the AC response of the current generated when a voltage is applied to the thin film as the fixed substrate approaches is detected.
  • FIG. 15 is a schematic cross-sectional view showing another example of a biomolecule analyzer.
  • the biomolecule analyzer of the present embodiment includes a pair of individual tanks having no nanopores at the end of the nanopore substrate 111. That is, in addition to the individual tank 105 for measuring biomolecules having the nanopores 110, there is a set of individual tanks 105A and 105B in which nanopores are not formed on the exposed thin film.
  • the individual tank 105A is provided in a region where the peripheral edge of the nanopore substrate and the fixed substrate 117A do not approach, and the individual tank 105B is provided in a region where the peripheral edge of the nanopore substrate and the fixed substrate 117A approach.
  • AC power sources 141A and 141B and ammeters 142A and 142B are connected to the individual electrodes arranged in the individual tanks 105A and 105B, respectively.
  • the difference between the currents measured by the ammeters 142A and 142B is sent to the impedance characteristic monitor system 140, and the amount of phase change is monitored by the computer 130.
  • the AC voltage to be applied be applied within a range that does not apply excessive stress to the thin film. This is because there is a possibility that a hole is formed in the thin film and the diameter of the already formed nanopore is enlarged by the application of the AC voltage. As a result, the impedance fluctuates and the approach detection accuracy of the fixed board may be deteriorated. Since the standard of the withstand voltage of a general semiconductor insulating film is 1 V / 1 nm, it is desirable that the applied AC voltage be 0.1 V / 1 nm or less in order to prevent excessive stress on the thin film.
  • the AC frequency F to be applied can be determined according to the capacitance C 0 between the counter electrodes, the solution resistance R 1 that changes according to the approach of the fixed substrate, and the solution resistance R 0 that does not depend on the approach of the fixed substrate.
  • FIG. 16 is a diagram showing the result of simulating the phase change of the current with respect to the solution resistance R 1 using the frequency F of the AC voltage to be applied as a parameter.
  • the capacitance C 0 is 1 nF and the solution resistance R 0 is 5 k ⁇ .
  • the resistance of the solution is 1 k ⁇ , and the resistance derived from the narrow space formed by the fixed substrate 117A and the nanopore substrate 111 is about 10 k ⁇ , it is necessary to detect 1 k ⁇ to 10 k ⁇ to detect the completion of the approach.
  • a frequency of about 10 kHz to 20 kHz may be selected based on FIG. This is because when the applied voltage is 10 kHz to 20 kHz, the amount of phase change when the solution resistance R 1 changes from 1 k ⁇ to 10 k ⁇ is relatively large.
  • the AC response through the thin film of the individual tank 105A to which the fixed substrate 117A is not approaching is monitored as a reference, and the difference between them is monitored.
  • the set of individual tanks 105A and 105B in which the nanopores are not formed in the thin film is provided only in one place of the nanopore substrate 111.
  • the set of the individual tanks 105A and 105B is 3 of the nanopore substrate 111. It is provided at more than one place, preferably at the four corners.
  • V 100 mV pp and frequency of 40 kHz
  • FIG. 17 is a schematic cross-sectional view showing another example of a biomolecule analyzer.
  • the biomolecule analyzer of the present embodiment is adjacent to three or more, preferably four corners, as proximity sensors in the peripheral area of the nanopore substrate 111 where the fixing member 117 approaches. Electrodes 118A and 118B were provided. A power source 125 and an ammeter 126 are connected to the adjacent electrodes 118A and 118B, and the ammeter 126 is connected to the computer 130 to analyze the detection signal.
  • adjacent electrodes are formed on the upper thin film fixing member 111B formed on the nanopore substrate 111 at the end of the region where the fixing substrate 117A approaches and at least three locations.
  • a voltage is applied from a separately provided power source 125 between adjacent electrodes including the pair of electrodes 118A and 118B, and an amount of current corresponding to a resistance value between the adjacent electrodes is monitored by an ammeter 126. Since the resistance value between the adjacent electrodes changes as the fixed substrate 117A approaches the adjacent electrode, it is possible to check whether the fixed substrate 117A is approaching or not approaching based on the amount of current detected by the ammeter 126. Become.
  • the distance between the fixed substrate 117A and the nanopore substrate 111 is 7 ⁇ m or more. Although there is almost no h dependency, it was found that there is a correlation between the distance h and the current decrease amount below that. Therefore, the height of the fixing member 117 can be adjusted by acquiring the correlation between the distance h and the current amount.
  • the present invention is not limited to the above-described embodiments, and includes various modifications.
  • the above-described embodiments have been described in detail in order to explain the present invention in an easy-to-understand manner, and it is not necessary to provide all the configurations described.
  • a part of a certain embodiment can be replaced with the configuration of another embodiment.
  • the configuration of another embodiment can be added to the configuration of a certain embodiment.
  • a part of the configuration of each embodiment can be added, deleted, or replaced with a part of the configuration of another embodiment.
  • Biomolecule analysis device 104 Common tank 105 Individual tank 110 Nanopore 111 Nanopore substrate 111A Thin film 111B Thin film fixing member 111C Lower thin film fixing member 113 Electrode substrate 116 DNA chain 117 Biomolecule fixing member 117A Biomolecule fixing substrate 117B Drive mechanism connecting member 117C Inclination correction mechanism 120 Power supply 121 Ammeter 130 Computer 201 Drive mechanism 502 Through hole

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Abstract

La présente invention concerne un dispositif d'analyse biomoléculaire qui comprend : un substrat nanoporeux (111) pourvu d'un film mince (111A) dans lequel une pluralité de nanopores (110) sont formés ; un réservoir commun (104) disposé sur la partie supérieure du substrat nanoporeux ; une pluralité de réservoirs individuels (105) disposés sur la partie inférieure du substrat nanoporeux, chacun des réservoirs individuels communiquant avec le réservoir commun par l'intermédiaire de nanopores ; une électrode commune (115A) disposée dans le réservoir commun ; un substrat d'électrode (113) sur lequel sont formées une pluralité d'électrodes individuelles, chacune d'entre elles étant disposée dans l'un de la pluralité de réservoirs individuels ; un élément de fixation de biomolécule (117) qui fixe les biomolécules et entraîne les biomolécules vers le substrat nanoporeux à l'intérieur du réservoir commun ; et un mécanisme de correction d'inclinaison (117C) disposé au-dessous de l'élément de fixation de biomolécule ou du substrat d'électrode. Le mécanisme de correction d'inclinaison se déforme en raison de la pression appliquée générée lorsque l'élément de fixation de biomolécule presse contre le substrat nanoporeux, et maintient la forme déformée.
PCT/JP2017/003456 2017-01-31 2017-01-31 Dispositif d'analyse biomoléculaire et élément de fixation de biomolécule Ceased WO2018142480A1 (fr)

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