WO2015152003A1 - 孔形成方法及び測定装置 - Google Patents
孔形成方法及び測定装置 Download PDFInfo
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- WO2015152003A1 WO2015152003A1 PCT/JP2015/059424 JP2015059424W WO2015152003A1 WO 2015152003 A1 WO2015152003 A1 WO 2015152003A1 JP 2015059424 W JP2015059424 W JP 2015059424W WO 2015152003 A1 WO2015152003 A1 WO 2015152003A1
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44791—Microapparatus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
Definitions
- the present invention relates to nanopore technology for measuring biomolecules, particularly DNA.
- Non-Patent Document 1 a TEM (Transmission Electron Microscope) apparatus is used as a nanopore forming method, and a membrane using a silicon nitride film (Si 3 N 4 ) or the like is irradiated with a reduced electron beam diameter to obtain energy and current. It is described that pores having a diameter of 10 nm or less are formed by controlling the above.
- TEM Transmission Electron Microscope
- Non-Patent Document 2 discloses another method for forming nanopores.
- a 10 nm thick non-porous membrane Si 3 N 4 film
- an aqueous solution potassium chloride
- the electrodes are placed in the KCl aqueous solution in the upper and lower chambers, respectively.
- aqueous solution potassium chloride
- the electrodes are placed in the KCl aqueous solution in the upper and lower chambers, respectively.
- voltage application is stopped when the current in the direction through the membrane exceeds a certain current threshold.
- 5 V is continuously applied between the electrodes, and the current between the electrodes reaches the current threshold between 400 s and 500 s, and the voltage application is stopped there.
- Nanopores having a pore diameter of about 5 nm are formed.
- a hole (nanopore 103) having a size on the order of nm comparable to the thickness of the DNA molecule 102 is provided in the region 101 of the thin film membrane, and the chamber 104 above / below the thin film membrane is filled with the aqueous solution 105.
- the DNA 102 to be measured is placed in one of the upper / lower chambers, and measurement is performed when the DNA passes through the nanopore to determine the structural characteristics and base sequence of the DNA (see FIG. 1).
- an optical measurement method photoexcitation 108 to measure the optical signal 109
- an electrical measurement method electrical measurement method
- Non-Patent Document 1 Using the TEM device drilling method of Non-Patent Document 1 and the non-patent document 2 voltage-drilling drilling method, we investigated the formation of nanopores used for optical measurement and tunnel current measurement. This will be described below.
- a conductor thin film 402 is formed on an insulating film membrane 401 and a hole 403 is formed in the conductor thin film 402 (hole array), or a bowtie 404 (Bowtie array) in which two conductor dots are arranged close to each other.
- the near-field light locally becomes strong only in the 5 nm region of the gap. Therefore, when the position where the DNA hole passes through the hole and the DNA molecule passes through the hole is not fixed, or when the hole position is deviated from the midpoint position of the gap, the excitation light received by the molecule becomes weak. A significant decline is inevitable. In principle, it is considered that the effect of the pore position itself on the displacement is larger than the case where the pore diameter is enlarged and the DNA passage position is not fixed.
- the pore diameter is (at the same time, for example, the tip of the bowtie is scraped off and the gap widens (from the original 5 nm to 7 nm), and the optical signal intensity is lowered.
- the hole diameter or hole position varies by only a few nanometers, the signal strength obtained changes by an order of magnitude. It is difficult to produce an optical measurement type nanopore device with uniform performance by drilling with TEM with good reproducibility.
- a tunnel current measurement method as another typical example of a device for measuring a molecule passing through a nanopore by providing a structure near the nanopore.
- a tunnel current measurement electrode pair 501 is provided on the plane of the insulating film membrane (FIG. 5A), and a hole is provided in the gap 502 between the tip portions of the electrode pair.
- a tunneling current flowing through the DNA molecule when the DNA molecule passes through the hole is measured while a voltage is applied thereto.
- the point of processing to open a hole aiming at the gap between a pair of conductor structures is the same as that of the aforementioned Bowtie.
- electrical wiring 503 is applied to each of the two dots of Bowtie and connected to a power source. It can be regarded as a device that is different from that used for measurement (FIG. 5B).
- the hole diameter has a variation of about 2 nm at 3 ⁇ with respect to the target 5 nm
- the hole position has a misalignment (position variation) of about 5 nm at 3 ⁇ from the midpoint of the target gap. .
- the change of the tunnel current when the DNA molecule passes through the nanopore is sharply weakened even if the DNA molecule deviates even a little from the current path connecting between the pair of electrodes arranged in the immediate vicinity of the nanopore.
- the gap is rapidly weakened. For this reason, even in the fabrication of nanopore devices used in the tunnel current measurement method, it is considered that the signal intensity obtained in subsequent measurements has changed by orders of magnitude, with only a few nanometers of hole diameter and hole position. .
- the drilling method using the TEM apparatus has not provided sufficient processing accuracy for measurement application. Furthermore, in the method using the TEM apparatus, not only the accuracy but also the apparatus cost is high and the throughput is low.
- the non-patent document 2 drilling method was tried next.
- the power supply device is less expensive than the method using the TEM device, and the vacuum system is not used, so that the throughput can be expected to be improved significantly.
- a membrane Si 3 N 4 film having a thickness of 10 nm is prepared, and a potassium chloride aqueous solution (KCl aqueous solution) is filled up and down across the top and bottom.
- the electrode was immersed in a KCl aqueous solution in the chamber, and a voltage was applied between both electrodes (FIG. 2).
- the process was stopped because the current value exceeded the threshold value in 440 seconds.
- a nanopore having a pore diameter of 5 nm could be formed in the first time at a position shifted about 40 nm to the left from the center of the membrane region.
- Non-Patent Document 2 With respect to the position of the hole, there is no straight hole at a position near the center of the membrane with respect to a flat membrane in which no characteristic structure is arranged. It was.
- the second time (second chip) has a hole shifted by 35 nm to the upper right from the center of the membrane, the third time is close to the center, and the fourth time, a hole is formed at a distance of about 45 nm to the lower right, licking from the central position. It was.
- the nanopore used in the optical measurement method and the tunnel current method needs to be formed at the position of the Bowtie gap or the gap of the electrode pair.
- a device in which a bowtie 404 for an optical measurement method is formed at the center of a membrane region 101 having a thickness of 10 nm and a device in which a gap structure of an electrode pair for a tunnel current measurement method is formed are prepared and loaded into a chamber 104 Then, the aqueous solution 105 was injected, and a hole was made by applying a voltage.
- a plasmon enhancement device such as Bowtie is an element that enhances near-field light, so that there may be some influence of light in an experiment in a bright place. Therefore, in the drilling process for these bowtie and electrode pair, a drilling experiment was conducted in a dark room (shield box) in order to eliminate the influence of light and confirm the effect of voltage application.
- nanopores 103 having a pore diameter of 5 nm were accurately formed at random locations unrelated to the arrangement of the structures. There was no effect of the structure formed on the membrane on the position of the hole formation (FIG. 6).
- the Bowtie structure used for the optical measurement method and the electrode pair used for the tunnel current method are formed on the surface of the membrane, and the hole making method by the TEM device of Non-Patent Document 1 and the hole by voltage application of Non-Patent Document 2 We examined the opening method.
- the hole drilling method using the TEM apparatus due to drift, a hole diameter variation of about 2 nm (3 ⁇ ) and a hole misalignment of about 5 nm (3 ⁇ ) occurred.
- the hole drilling method by applying voltage the variation in hole diameter is improved to 1 nm (3 ⁇ ), and the cost and high throughput can be expected.
- a membrane, bowtie or electrode pair with no structure is formed on the surface.
- the drilling position was not determined.
- the blocking current method shown in Non-Patent Document 2 the membrane does not have a structure other than nanopores, and the blocking of molecules with ion current flowing through the nanopores is measured
- the position of the holes need not be determined accurately.
- the optical measurement method using near-field light with the plasmon enhancement structure and the tunnel current method using the gap between the electrode pairs not only the hole diameter is reduced and the accuracy is improved, but also at a fixed position of the structure. Position control to drill holes is necessary.
- the insulating film on which the near-field light generating element is mounted is irradiated with light in the electrolytic solution, or the film is irradiated with light.
- the second step of detecting the value is repeated, and the procedure of repeating the first step and the second step is stopped when the current value reaches or exceeds a preset threshold value.
- the measuring apparatus of the present invention includes a light source for irradiating light to an insulating film on which a near-field light generating element is mounted, a mechanism for installing the film in the chamber, and an electrolyte solution in the chamber in which the film is installed.
- an introduction port for introducing a substance to be measured, a first electrode and a second electrode provided across a film, a power source for applying a voltage between the first electrode and the second electrode, and a voltage
- An ammeter that detects the current value obtained by applying the light source, a control unit that controls the light source and the power source, and a storage unit that stores the relationship between the size of the hole formed by applying a voltage to the film and the current value
- the control unit applies a first voltage between the first electrode and the second electrode while irradiating the film with light or after irradiation, and after applying the first voltage, the first voltage is applied to the film.
- the measurement target substance such as a biomolecule
- the measurement target substance that passes through the formed hole or a label illuminator added to the measurement target substance detects light emitted by receiving light from the light source. It has the photodetector provided with the color identification mechanism, and the memory
- the storage unit stores a value for each substance constituting the measurement target substance.
- an insulating film in which an electrode pair is placed with a gap interposed therebetween is placed in the electrolyte solution, and a voltage is applied to the electrode pair or after a voltage is applied, the film is sandwiched.
- the measuring apparatus of the present invention includes a mechanism for installing an insulating film in which an electrode pair is placed with a gap in between in a chamber, and an inlet for introducing an electrolyte and a substance to be measured into the chamber in which the film is installed.
- a first ammeter for detecting a current value obtained by applying a voltage from the first power supply
- a controller for controlling the first and second power supplies, and applying a voltage to the film
- a storage unit that stores the relationship between the size of the hole formed by the current and the current value, and the control unit applies the voltage from the second power source to the electrode pair or after applying the voltage to the electrode pair.
- a first voltage is applied between the second electrode and the first electrode after application of the first voltage.
- Control that detects a current value flowing between the first electrode and the second electrode by applying the second voltage between the two electrodes and the first ammeter by applying the second voltage Is repeated, and when the current value is stored in the storage unit and reaches or exceeds a preset threshold value, it is stopped to repeat the control as a hole is formed in the film.
- the measurement unit further includes a second ammeter that detects a current value flowing through the electrode pair as the measurement target substance passes through the formed hole. A value for each substance constituting the measurement target substance is stored.
- the effects obtained by typical ones are as follows.
- a hole can be formed at a position suitable for measurement.
- the hole size can be controlled at an angstrom level while monitoring with high accuracy. Thereby, measurement with good reproducibility becomes possible using the nanopore of the optical measurement method or the tunnel current measurement method. This is a simple method and can be expected to reduce costs.
- a plasmon enhancing structure is provided on a membrane
- the plasmon enhancing structure is a Bowtie structure as an example, and a hole is accurately formed in a gap of the Bowtie structure.
- FIG. 7 shows the entire apparatus.
- the chamber 104 is provided with an aqueous solution inlet 106 and an outlet 107 for introducing an aqueous solution 105 containing an electrolytic solution and the like, and an insulating film 101 is sandwiched between the electrode 1 and the electrode 2. It has a mechanism to do. From the aqueous solution inlet 106, an electrolytic solution, a substance to be measured, and the like can be introduced into the chamber 104.
- a near-field light generating element 404 that generates near-field light when irradiated with light as shown in FIG. 4 is placed on the insulating film 101.
- This apparatus includes a light source 203 that emits light to the near-field light generating element 404.
- a power source 1 (201) for applying a voltage between the electrode 1 and the electrode 2, and a current for detecting a current flowing between the first electrode and the second electrode when the voltage is applied It has a total of 1 (202) and has a control unit 701 for controlling them.
- the control unit 701 includes a processor, a memory, and the like necessary for controlling a power source, a light source, and the like.
- a chip formed with a Bowtie structure 404 in which two conductive dots having a size of about 40 nm are arranged in proximity to each other on a Si 3 N 4 membrane 101 having no hole with a thickness of 10 nm is loaded into the chamber and separated by the membrane.
- the KCl aqueous solution 105 was filled on both sides of the chamber, and the electrodes 1 and 2 were immersed in the KCl aqueous solution in the chambers on both sides.
- a voltage of 4 V was first applied between the electrode 1 and the electrode 2 by the power source 1 for 2 minutes in a dark environment where the chamber 104 was shielded by a shield box.
- the current value that was 800 pA (corresponding to a pore diameter of 2 nm) immediately after application of a voltage of 4 V for measurement increased to 1060 pA (corresponding to a pore diameter of 2.3 nm) 30 seconds after the start of application of the voltage for measurement. (FIG. 12). It is desirable that the hole does not expand during the period in which the voltage for measuring the diameter of the hole is applied. By applying a voltage in the liquid, a 2 nm hole could be formed in the first 2 minutes, but the voltage of 4 V applied for the subsequent pore diameter measurement was too high. Seems to have spread. Moreover, the position of the hole was formed at a position unrelated to the Bowtie structure formed on the membrane.
- the position of hole formation was not determined, but the following guidelines were obtained for the applied voltage and the application time.
- the Si 3 N 4 membrane having a thickness of 10 nm which is used for hole formation
- a voltage of 4 V when a voltage of 4 V is continuously applied, a hole is formed and the hole diameter gradually increases.
- ions when a voltage of 1 V is applied, ions are passed through the hole. A current flows, and the hole diameter can be estimated from the current value, but the hole diameter does not increase.
- the hole formation of about 2 nm is completed by repeating the voltage application 6 to 8 times. For example, when the applied voltage is made higher than 4 V while maintaining the thickness of the membrane, it is thinner than this.
- the amount of hole expansion by applying a voltage for 2 minutes at a time increases, and the desired hole diameter is reached at a smaller number of times. Further, even when the material is changed to a material having low insulation resistance, holes are easily formed with a small number of times. Thus, if the number of times decreases, it may not be possible to stop at the target hole diameter, and the target hole diameter may be exceeded. In such a case, measure the hole diameter with a voltage of 1V each time as the hole diameter expands in small increments, such as by changing the voltage application for drilling that was in 2 minute increments to 1 minute increments. By proceeding while confirming, it is possible to more accurately match the target hole diameter.
- the hole diameter measurement time was fixed at 30 seconds. However, when the voltage is lowered to 1V, the hole does not expand during the hole diameter measurement. It doesn't matter. In this example, noise is present in the ion current being measured, and the error in the pore diameter was large at 10% or more in the measurement for several seconds. However, by measuring the current for 30 seconds and averaging the noise, The hole diameter could be estimated with an error of less than%.
- the light source 1 is installed in the shield box, turned on, and the plasmon enhancement structure (Bowtie structure 404) formed on the membrane 101 is irradiated with light to generate near-field light at the Bowtie gap position.
- a laser having a wavelength of 785 nm and an output of 50 mW was used as the light source.
- the electrodes 1 and 2 were immersed in the KCl aqueous solution 105 in the chambers 104 on both sides separated by the membrane (FIG. 7), and a voltage of 4 V was applied between the electrodes by the power source 1 for 2 minutes. I tried drilling.
- the light source 1 was turned off, a voltage of 1 V was applied between the electrodes for 30 seconds, and the current value was measured accurately.
- the current value when a voltage of 4 V for drilling was applied increased to 800 pA (corresponding to a hole diameter of 2 nm), and it was confirmed that there was a hole.
- the current flowing through the hole is stable at 200 pA (corresponding to a hole diameter of 2.0 nm) for 30 seconds without being changed (FIG. 14).
- the current flowing through the hole may be measured by applying a voltage of 1 V between the electrode 1 and the electrode 2 by the power source 1 without illuminating the light source without turning off the light source.
- the value is inaccurate due to the effect of charge-up due to light, etc. Therefore, it is better to turn off the light source when applying the hole diameter measurement voltage.
- a hole is formed by applying a voltage while irradiating light.
- light is irradiated in advance to the near-field light generating element on the membrane, and light is emitted in the vicinity of the near-field light generating element.
- a membrane is placed in the electrolyte solution of the chamber and voltage is applied to form a hole.
- the laser beam intensity was increased by a factor of 10 to a chip in which a Bowtie structure was formed on a Si 3 N 4 membrane having a thickness of 10 nm.
- Two experiments were conducted: an experiment in which the drilling voltage was lowered to 2 V, and an experiment in which the laser beam wavelength was changed to 638 nm, which is a short wavelength, and the drilling voltage was lowered to 2 V. The voltage was lowered to 2 V. In spite of this, it was found that there was a hole in the Bowtie gap position. This is probably due to the enhancement of the excitation density at the Bowtie gap position or excitation to a high energy level.
- the hole diameter measurement voltage for 30 seconds after applying the drilling voltage for 2 minutes at 2 V is set to 2 V, which is the same as the hole drilling voltage, for simplicity, the laser beam is measured during the hole diameter measurement. It was also found that the pore diameter did not expand in 30 seconds if was turned off.
- the voltage at the time of drilling and the hole diameter measurement is simply the same except that the voltage is lowered at the time of hole diameter measurement. It has also been found that by simply turning off the light irradiation during measurement, it is possible to set desired drilling conditions that do not cause the hole to expand during hole diameter measurement.
- FIG. 8 shows the entire apparatus, and a description of the same parts as in FIG. 7 is omitted.
- the insulating film 101 includes an electrode pair 501 and a power source 2 (204) for applying a voltage to the electrode pair 501.
- a hole is formed in the gap of the electrode pair 501 by applying the voltage application method as described in the first embodiment while applying a voltage to the electrode pair 501 or after applying the voltage. Specific examples are shown below.
- An electrode pair 501 was formed on a Si 3 N 4 membrane having a thickness of 10 nm and no holes, and a chip having a gap 502 of 3 nm at a position where the tips of the electrode pair faced was loaded into the chamber.
- a power source 2 for applying a voltage to the electrode pair 501 was connected. Both sides of the chamber isolated by the membrane were filled with KCl aqueous solution, and electrodes 1 and 2 were immersed in the KCl aqueous solution in the chambers on both sides, respectively, and a power source 1 was connected thereto. Since it is different from the photoexcitation device shown in Example 1, it may not be affected by light. However, the same shield box used in Example 1 was used for comparison by forming a hole in the membrane in the same experimental environment. The experiment was carried out in a dark environment with light shielding.
- a voltage of 4 V was applied by the power source 1 between the electrodes 1 and 2 for 2 minutes without applying a voltage (power source 2) to the electrode pair for tunnel current measurement.
- a voltage of 1 V was applied between both electrodes for 30 seconds, and the current value was accurately measured.
- a voltage of 2 V was applied to the tunnel current measurement electrode pair 501 by the power source 2 (204), and a voltage was applied to the 3 nm gap 502 at the tip of the electrode pair on the membrane without a hole. Since the KCl aqueous solution is filled between the gaps, a current flows through the gap 502 of the electrode pair. In this situation, the electrodes 1 and 2 were immersed in the KCl aqueous solution in the chambers on both sides separated by the membrane, respectively, and the power supply 1 applied a voltage of 4 V between the electrodes for 2 minutes to try to make a hole. .
- the voltage applied to the gap at the tip of the tunnel current measuring electrode pair 501 is turned off, and a voltage of 1 V is applied between the electrodes 1 and 2 by the power source 1. It was applied for 30 seconds and the current value was accurately measured. As a result, it was confirmed that pores were formed almost unchanged for 30 seconds at 250 pA.
- the vicinity of the gap at the tip of the electrode pair of the tunnel current measuring electrode pair was carefully observed to find the position where the hole was formed. As a result, a hole close to an ellipse having a major axis of 2.2 nm and a minor axis of 2.0 nm was found. It was confirmed that it was formed at the gap position of the part.
- the shape of the hole is slightly extended between the tips of the tunnel current measuring electrode pair in the direction of the tips of both electrodes. Since a current is induced in the gap at the electrode tip by the voltage applied by the power source 2, the electronic excitation state and the charged state of the membrane material are changed, and dielectric breakdown is likely to occur. It is thought that a hole was formed in the surface.
- Example 1 and Example 2 a specific position on the membrane was excited by a functional structure capable of realizing light enhancement, local current excitation, and the like, so that the drilling position could be determined.
- a functional structure capable of realizing light enhancement, local current excitation, and the like so that the drilling position could be determined.
- a bowtie as a plasmon enhancement structure on a membrane and one having a tunnel current measurement electrode pair on a membrane as an example, one having a bowtie as a plasmon enhancement structure on a membrane Is used to investigate the relationship between the drilling voltage, the number of voltage applications, and the time (throughput) required to complete the hole.
- Both sides were filled with a KCl aqueous solution, and electrodes 1 and 2 were immersed in the KCl aqueous solution in the chambers on both sides.
- the goal was to make a 3 nm hole.
- the procedure 1 was performed 10 times, and when the current at the time of applying 1V for the hole diameter measurement reached 310 pA, the processing was switched to the processing in which the applied voltage was increased (procedure 2). From the 11th voltage application, the procedure for increasing the applied voltage setting for drilling from 4V to 5V and then measuring the hole diameter at 1V (procedure 2) was performed 6 times (up to the 16th in total). However, it showed a tendency to increase from 310 pA as shown in the 11th to 16th times of FIG. 17, and it was judged that 450 pA (hole diameter of 3 nm) was reached when the voltage of 1 V for the 16th hole diameter measurement was applied.
- the expansion of the hole tends to saturate as the number of times increases, and it is large from the beginning when it is desired to expand the hole.
- the desired hole diameter is not easily reached even if the number of times is increased. It was found that when it is desired to increase the hole diameter or to form a hole having a slightly larger diameter, the time required to complete the hole can be shortened by increasing the drilling voltage. However, if the voltage is increased too much from the beginning, there is a concern that the desired hole diameter may be exceeded only by performing procedure 1 a few times. Once the hole has been expanded too much, it cannot be narrowed again, so it is better to aim at a smaller size, switch the voltage on the way, and expand it a little to complete.
- FIG. 18 (a) shows the current change when forming a 2 nm diameter hole in a 10 nm thick membrane
- FIG. 18 (b) shows the current change when forming a 3 nm diameter hole in a 10 nm thick membrane
- FIG. 18 (c) shows a current change when the drilling is further performed with the aim of forming a hole having a diameter of 4 nm in the membrane having a thickness of 20 nm by increasing the load of the drilling process.
- a reverse voltage of ⁇ 2 V is set to 5 immediately after drilling under the conditions of FIG. 18C (drilling voltage 8.5 V, hole diameter measuring voltage 1 V). It applied for 2 second, and it changed into the conditions which transfer to a hole diameter measurement after that.
- a pore diameter measurement voltage of 1 V was applied, the current value once fluctuated below the graph, and then no behavior was observed that settled to a constant value, and the current during the pore diameter measurement became a constant value.
- FIG. 20 shows a state in which the charge accumulated on the surface is eliminated by applying a reverse voltage when there is an influence of charging due to the hole punching voltage, and the hole diameter can be measured with high accuracy.
- a light source 1 (203) and a photodetector 1 (206) are used to irradiate the bowtie of the near-field light generating element 404 from the light source, and from the DNA molecules passing through the holes. Is measured at the detector.
- a chip in which 100 elements of 10 ⁇ 10 elements each having a bowtie structure for an optical measurement system formed on a 5 nm-thick membrane is formed in parallel and separated by a membrane.
- Each side of the chamber was filled with a KCl aqueous solution, and the electrodes 1 and 2 were immersed in the KCl aqueous solution in the chambers on both sides.
- 100 electrodes are arranged in the upper region, one for each of the 100 parallel elements, while the lower side of the membrane Since the electrode 2 in the region may be at a common potential, it is a common electrode for 100 membranes.
- 100 elements were irradiated with light, and a voltage was applied to each one element by the method described above to form holes.
- one switch 505 is provided for each of 100 electrodes 1 for 100 membranes. 100 holes were formed in each Bowtie gap position while applying voltage in order and checking the hole diameter one by one. Thereafter, without removing the membrane chip from the chamber, the aqueous solution containing the DNA was continuously injected into the upper chamber to shift to the measurement of DNA molecules.
- mixed light of laser beams having wavelengths of 505 nm and 642 nm was used as light for exciting the fluorescent dye for analyzing the four types of bases (A, G, C, T) of DNA.
- This is one of the general wavelength selections for emitting all of the four types of fluorescence shown below. Since the intensity of the excitation light is enhanced by the plasmon enhancement device, depending on the enhancement, in this experiment, it was sufficient for the laser output to have a wavelength of about 50 mW for both the wavelength of 505 nm and the wavelength of 642 nm.
- a non-coherent light source may be used instead of a laser as long as it can output light having the same excitation wavelength.
- A is 520 nm as the wavelength for analysis.
- 666 nm for C is 520 nm as the wavelength for analysis.
- 567 nm for C is a nm as the wavelength for analysis.
- 702 nm for T is a nm as the wavelength for analysis.
- FIG. 22B shows a signal analyzed using a color filter plate for the color selection function.
- a spectroscopic unit using a prism or a diffraction grating may be used as long as four types can be identified.
- results of G, A, G, T, C, and T were obtained.
- the analysis unit performs computing such as duplication characteristics and estimation from the superposition of a plurality of data, and by combining the analyzed fragments, It was possible to clarify the order of arrangement, and the basic functions as a DNA sequencer could be configured and confirmed.
- peaks having a wavelength difference little by little peaks having a difference of about several hundred cm -1 at the maximum in terms of energy
- the vibrational spectrum structure Due to the difference in the molecular structure, the vibrational spectrum structure is different, and this appears as a difference in the peak wavelength of the spectrum in the observation wavelength region in Raman spectroscopy. In the field of Raman spectroscopy, this is called Raman shift. That is, since the four types of bases have different Raman shifts, they appear as separated peaks that can identify the four types of bases on the Raman spectrum.
- FIG. 23A shows a schematic block diagram in the case of using Raman spectroscopy.
- the spectrum obtained when excited at a wavelength of 638 nm using a diffraction grating spectroscope and an optical system is imaged in the color selection function portion. 23 (b).
- the peak wavelengths can be separated in this way, the results of trying the DNA analysis using these four peaks are shown in FIG.
- FIG. 23C the results with A, T, G, C, T, and A were obtained.
- Such data information is collected, and the analysis unit performs computing such as duplication characteristics and estimation from the overlapping of a plurality of data.
- the order could be clarified, and the basic function as a DNA sequencer could be constructed and confirmed.
- the current flowing through the nanopore when the voltage is applied to the electrodes above and below the membrane without using the light source 1 (203) and the light detector 1 (206) among the ones shown in FIG. 9 is the ammeter 1 (202). Measure the change in current as DNA molecules pass through while monitoring at. This is a method of measuring the difference in the blocking rate at which DNA molecules block the nanopore and identifying the type of base that has passed through.
- the configuration shown in FIG. 11 will be described in which the light source 1 (203), the photodetector (206), and the optical system (lens, color identification) associated therewith are not used.
- a chip in which 100 ⁇ 10 elements having a Bowtie structure for an optical measurement system formed on a 5 nm-thick membrane is formed in parallel is loaded into a chamber, and both sides of the chamber isolated by the membrane are filled with a KCl aqueous solution.
- the electrodes 1 and 2 were immersed in the KCl aqueous solution in the chambers on both sides.
- 100 electrodes are arranged in the upper region, one for each of the 100 parallel elements, while the lower side of the membrane Since the electrode 2 in the region may be at a common potential, it is a common electrode for 100 membranes.
- 100 elements were irradiated with light, and a voltage of 2 nm was formed by applying a voltage to the one element per element by the method described above.
- a voltage one by one with a switch provided on each of the 100 electrodes 1 for 100 membranes and checking the hole diameter one by one, 100 holes were formed at each Bowtie gap position. Thereafter, without removing the membrane chip from the chamber, the aqueous solution containing the DNA was continuously injected into the upper chamber to shift to the measurement of DNA molecules.
- DNA molecules are not measured by optical measurement, but the amount of current flowing through the hole is measured by blocking the hole when the molecule passes through the hole (blocking).
- Current method First, with the light source 1 turned off, a voltage was applied to the electrode 1 and the electrode 2 by the power source 1 and the ion current flowing through the hole was continuously monitored by an ammeter attached to the power source 1; When passing through, a characteristic decrease (blocking) of the ionic current was observed, and it was possible to capture how DNA molecules in the aqueous solution passed through the pores.
- a chip in which 100 pairs of 10 ⁇ 10 elements each having an electrode pair for tunnel current measurement formed on a 5 nm-thick membrane is formed in a chamber and separated by the membrane.
- Each side of the chamber was filled with a KCl aqueous solution, and the electrodes 1 and 2 were immersed in the KCl aqueous solution in the chambers on both sides.
- 100 electrodes are arranged in the upper region, one for each of the 100 parallel elements, while the lower side of the membrane
- the electrode 2 in the region is a common electrode for 100 membranes.
- a voltage is applied to the electrode pair for tunnel current measurement of 100 elements by the power source 2 to excite the gap of the electrode pair, and a voltage is applied to the one element per element by the power source 1 by the above-described method. Formed.
- one switch 505 is provided for each of 100 electrodes 1 for 100 membranes. 100 holes were formed in the gap position of each electrode pair while applying voltage in order and checking the hole diameter one by one. Thereafter, without removing the membrane chip from the chamber, the aqueous solution containing the DNA was continuously injected into the upper chamber to shift to the measurement of DNA molecules.
- a voltage was applied from the power source 2 to the electrode pair on the membrane, and when the DNA molecules passed through the holes, the tunnel currents flowing through the respective bases were measured. It was found that the tunneling currents flowing through the four different base structures of the DNA molecules were different, and that the DNA base sequence could be obtained by this method.
- the hole position is aligned with the electrode gap so that a tunnel current can flow through the molecule by the method described in this specification, and the hole diameter is controlled with high accuracy. The first measurement was realized.
- the nanopore formation method using the method of Example 1-7 is not limited to the SiON membrane, the SiO 2 membrane, the alumina membrane, the HfO 2 membrane, the HfSiON membrane, the TiO 2 membrane, the zirconia membrane, the ZrSiO 4 membrane, Application was also made to inorganic material membranes such as yttria membranes and other polymer membranes. Although the voltage required for drilling differs depending on the material due to differences in material characteristics such as band gap, it was confirmed that the present technology can be basically applied to the above materials.
- a common light source for near-field light generation and DNA analysis by optical measurement can be used as long as the power and wavelength can be varied so as to be suitable for measurement.
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Abstract
Description
102 DNA分子
103 ナノポア
104 チャンバ
105 水溶液
106 水溶液入口
107 水溶液出口
108 光励起
109 光信号
110 電気信号
201 電源1
202 電流計1
203 光源
204 電源2
205 電流計2
206 光検出器
401 絶縁膜メンブレン
402 導体薄膜
403 ホール
404 Bowtie
405 ギャップ
406 導体ドット
407 上下の導体ドット間の絶縁体薄膜
408 近接場光発生位置
501 電極対
502 電極対間のギャップ
503 電気配線
504 電流発生位置
505 選択スイッチ
701 制御ユニット
Claims (14)
- 膜に対し孔を形成する方法であって、
近接場光発生素子が載置された絶縁性の膜に対し、電解液中で前記膜に光を照射しながら、又は、前記膜に光を照射後に前記膜を前記電解液中に設けた後、前記電解液中で前記膜を挟んで設置された第1の電極と第2の電極との間に第1の電圧を印加する第1の工程と、
前記第1の工程後、前記第1の電極と前記第2の電極との間に第2の電圧を印加し、前記第2の電圧を印加することにより前記第1の電極と前記第2の電極との間に流れる電流値を検出する第2の工程と、を繰り返し、
前記第1の工程と前記第2の工程を繰り返す手順を、前記電流値が予め設定した閾値に到達又は超えた場合に止めることを特徴とする孔形成方法。 - 前記第2の電圧は前記第1の電圧よりも小さいことを特徴とする請求項1に記載の孔形成方法。
- 前記手順のn回目よりもm回目(但しn<m)の方が前記第1の電圧が大きくなるように又は前記第1の電圧の印加時間が長くなるように前記手順を行うことを特徴とする請求項1に記載の孔形成方法。
- 前記第1の工程と前記第2の工程の間に、前記第1の電圧とは逆極性の電圧を印加する工程を有することを特徴とする請求項1に記載の孔形成方法。
- 近接場光発生素子が載置された絶縁性の膜に対し光を照射する光源と、
前記膜をチャンバに設置する機構と、
前記膜が設置されたチャンバに電解液及び測定対象物質を導入する導入口と、
前記膜を挟んで設けられる第1の電極及び第2の電極と、
前記第1の電極及び前記第2の電極との間に電圧を印加する電源と、
前記電圧を印加することにより得られる電流値を検出する電流計と、
前記光源及び前記電源を制御する制御部と、
前記膜に電圧を印加することにより形成される孔の大きさと電流値との関係を記憶した記憶部とを備え、
前記制御部は、前記膜に対し光を照射しながら又は照射後に前記第1の電極と第2の電極との間に第1の電圧を印加し、前記第1の電圧の印加後に前記第1の電極と前記第2の電極との間に第2の電圧を印加し、前記第2の電圧を印加することにより前記第1の電極と前記第2の電極との間に流れる電流値を検出する制御を繰り返し行い、前記電流値が前記記憶部に記憶され予め設定した閾値に到達又は超えた場合に前記膜に孔が形成されたとして前記制御を繰り返すのを止めることを特徴とする測定装置。 - 形成された前記孔を通過する前記測定対象物質又は前記測定対象物質に付加された標識発光体が、前記光源からの光を受けて放出する光を検出する色識別機構を備えた光検出器を有し、前記記憶部は、前記測定対象物質を構成する物質毎の光検出値を記憶していることを特徴とする請求項5に記載の測定装置。
- 前記第1の電極と前記第2の電極との間に電圧を印加することにより、形成された前記孔を前記測定対象物質が通過するのに伴い流れる電流値について、前記記憶部は、前記測定対象物質を構成する物質毎の値を記憶していることを特徴とする請求項5に記載の測定装置。
- 前記絶縁性の膜は、シリコン窒化膜、シリコン酸化膜、シリコン酸窒化膜、アルミナ膜、ハフニウム酸化膜、ハフニウム酸窒化膜、HfSiON、チタン酸化膜、ジルコニア膜、ZrSiO4、イットリア膜、ポリマ膜のいずれかであることを特徴とする請求項5に記載の測定装置。
- 膜に対し孔を形成する方法であって、
電極対がギャップを挟んで載置された絶縁性の膜を電解液中に設置し、前記電極対に電圧を印加しながら、又は電圧を印加した後に、前記膜を挟んで設置された第1の電極と第2の電極との間に第1の電圧を印加する第1の工程と、
前記第1の工程後、前記第1の電極と前記第2の電極との間に第2の電圧を印加し、前記第2の電圧を印加することにより前記第1の電極と前記第2の電極との間に流れる電流値を検出する第2の工程と、を繰り返し、
前記第1の工程と前記第2の工程を繰り返す手順を、前記電流値が予め設定した閾値に到達又は超えた場合に止めることを特徴とする孔形成方法。 - 前記第2の電圧は前記第1の電圧よりも小さいことを特徴とする請求項9に記載の孔形成方法。
- 前記手順のn回目よりもm回目(但しn<m)の方が前記第1の電圧が大きくなるように又は前記第1の電圧の印加時間が長くなるように前記手順を行うことを特徴とする請求項9に記載の孔形成方法。
- 前記第1の工程と前記第2の工程の間に、前記第1の電圧とは逆極性の電圧を印加する工程を有することを特徴とする請求項9に記載の孔形成方法。
- 電極対がギャップを挟んで載置された絶縁性の膜をチャンバに設置する機構と、
前記膜が設置されたチャンバに電解液及び測定対象物質を導入する導入口と、
前記膜を挟んで設けられる第1の電極及び第2の電極と、
前記第1の電極と前記第2の電極との間に電圧を印加する第1の電源と、
前記電極対に電圧を印加する第2の電源と、
前記第1の電源により電圧を印加することにより得られる電流値を検出する第1の電流計と、
前記第1及び第2の電源を制御する制御部と、
前記膜に電圧を印加することにより形成される孔の大きさと前記電流値との関係を記憶した記憶部とを備え、
前記制御部は、前記電極対に対し前記第2の電源から電圧を印加しながら又は印加後に、前記第1の電極と第2の電極との間に第1の電圧を印加し、前記第1の電圧の印加後に前記第1の電極と前記第2の電極との間に第2の電圧を印加し、前記第2の電圧を印加することにより前記第1の電極と前記第2の電極との間に流れる電流値を前記第1の電流計により検出する制御を繰り返し行い、前記電流値が前記記憶部に記憶され予め設定した閾値に到達又は超えた場合に前記膜に孔が形成されたとして前記制御を繰り返すのを止めることを特徴とする測定装置。 - 形成された前記孔を前記測定対象物質が通過するのに伴い前記電極対に流れる電流値を検出する第2の電流計を有し、前記記憶部は、さらに前記電流値について前記測定対象物質を構成する物質毎の値を記憶していることを特徴とする請求項13に記載の測定装置。
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WO2018105123A1 (ja) * | 2016-12-09 | 2018-06-14 | 株式会社日立ハイテクノロジーズ | ナノポア形成方法、ナノポア形成装置及び生体分子計測装置 |
CN109890497A (zh) * | 2016-12-09 | 2019-06-14 | 株式会社日立高新技术 | 纳米孔隙形成方法、纳米孔隙形成装置以及生物分子测量装置 |
GB2570849B (en) * | 2016-12-09 | 2022-03-16 | Hitachi High Tech Corp | Nanopore-forming method, nanopore-forming device and biomolecule measurement device |
US11499959B2 (en) | 2016-12-09 | 2022-11-15 | Hitachi High-Tech Corporation | Nanopore-forming method, nanopore-forming device and biomolecule measurement device |
US10443146B2 (en) | 2017-03-30 | 2019-10-15 | Lam Research Corporation | Monitoring surface oxide on seed layers during electroplating |
US11208732B2 (en) | 2017-03-30 | 2021-12-28 | Lam Research Corporation | Monitoring surface oxide on seed layers during electroplating |
WO2018183755A1 (en) * | 2017-03-30 | 2018-10-04 | Lam Research Corporation | Monitoring surface oxide on seed layers during electroplating |
US11333623B2 (en) * | 2017-12-05 | 2022-05-17 | Hitachi High-Tech Corporation | Hole forming method and hole forming apparatus |
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US11181502B2 (en) | 2021-11-23 |
US20170138899A1 (en) | 2017-05-18 |
JP2015197385A (ja) | 2015-11-09 |
CN106133507A (zh) | 2016-11-16 |
DE112015001642B4 (de) | 2021-12-09 |
DE112015001642T5 (de) | 2016-12-29 |
JP6209122B2 (ja) | 2017-10-04 |
GB2538482A (en) | 2016-11-16 |
GB201616120D0 (en) | 2016-11-09 |
CN106133507B (zh) | 2018-06-15 |
GB2538482B (en) | 2020-08-26 |
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