WO2020105318A1 - Biomolecule analysis device and biomolecule analysis method - Google Patents

Abstract

The present invention makes it possible to suppress generation of background noise and measure a subject molecule with high SN ratio. This biomolecule analysis device includes: a thin film having nanopores; a pair of liquid vessels accommodating an electrolytic solution and disposed so as to sandwich the thin film; a pair of electrodes respectively disposed on the pair of liquid vessels; and a measurement unit connected to the pair of electrodes. The device is characterized in that the electrolytic solution has biomolecules modified by modifying molecules dissolved therein and has a pH of greater than 8.0, and the modifying molecules are prevented from passing through the nanopores on their own when the electrolytic solution has a pH of greater than 8.0.

Description

Biomolecule analyzer and biomolecule analysis method

The present disclosure relates to a biomolecule analysis device and a biomolecule analysis method.

-Technology using nanopore devices is being investigated as a means to detect and analyze molecules and particles present in aqueous solutions. The nanopore device is provided with pores (nanopores) of the same size as the molecules or particles to be detected in the membrane, fills the chambers above and below the membrane with an aqueous solution, and contacts the aqueous solution in both chambers with electrodes. Is provided. The detection target is introduced into one side of the chamber, passed through the nanopores by applying a potential difference between the electrodes and electrophoresed, and the time change of the ionic current (blocking signal) flowing between both electrodes is measured to detect the detection target. It is possible to detect the passage of an object and analyze the structural characteristics of the detection target.

In the production of nanopore devices, a method of forming a nanopore by processing a semiconductor substrate or a semiconductor material by a semiconductor process has attracted attention because of its high mechanical strength. As such a method for forming nanopores, for example, in Non-Patent Document 1, a silicon nitride film (SiNx film) is used as a membrane, and a TEM (transmission electron microscope) device is used to reduce the irradiation area of an electron beam to a small area on the membrane. It is disclosed that nanopores having a diameter of 10 nm or less are formed by controlling energy and current.

Further, Patent Document 1 and Non-Patent Documents 2 to 4 disclose nanopore forming methods utilizing dielectric breakdown of a membrane. In these methods, first, the upper and lower chambers sandwiching a non-perforated SiNx membrane are filled with an aqueous solution, the electrodes are immersed in the aqueous solution of each chamber, and a high voltage is continuously applied between both electrodes. When the current value between the electrodes suddenly rises (dielectric breakdown of the membrane) and the preset cutoff current value is reached, it is judged that the nanopores have been formed, and by stopping the application of high voltage, the nanopores are stopped. To form. The present nanopore forming method has advantages that the manufacturing cost is significantly reduced and the throughput is improved as compared with the nanopore formation using the TEM device. In addition, the present nanopore forming method can shift to measurement of the detection target without removing the membrane from the chamber after forming the nanopore in the membrane. Therefore, there is an advantage that the nanopore is not exposed to pollutants in the air and noise during measurement is reduced.

The purpose of measurement using nanopore is to detect and count the detection target in aqueous solution. For example, in Non-Patent Document 5, PNA and PEG are bound only to DNA having a specific sequence present in an aqueous solution, and a change in ionic current when DNA modified with PNA and PEG passes through a nanopore is measured. Discloses to detect and count DNA having a specific sequence.

In this way, in the nanopore measurement that detects and counts the detection target, the modification molecule is bound to the detection target and the amount of blocking signal and the difference in the blocking time are measured to perform detection and quantification.

Another application of measurement using nanopores is decoding the base sequence of DNA (DNA sequencing). That is, it is a method of determining the sequence of four types of bases in a DNA chain by detecting a change in ionic current when DNA passes through a nanopore.

International Publication No. 2013/167955

However, as in the above-mentioned conventional example, in the nanopore measurement for detecting and counting the detection target using the nanopore, when the unbound modified molecule alone passes through the nanopore, the signal derived from the modified molecule becomes the background. If the size of the unmodified measurement target molecule is close to that of the modified molecule, or if the size of the modified measurement target molecule is close to the size of the modified molecule, the difference between these signals will be recognized and modified during analysis. It becomes difficult to remove the signal derived from the molecule.

Therefore, the present disclosure provides a biomolecule analysis device and a biomolecule analysis method for suppressing generation of background noise and measuring a target molecule with a high SN ratio.

The biomolecule analysis device of the present disclosure is a thin film having nanopores, a pair of liquid tanks arranged so as to sandwich the thin film, and a pair of liquid tanks containing an electrolyte solution, and a pair of electrodes arranged in each of the pair of liquid tanks, A measuring unit connected to the pair of electrodes, wherein the electrolyte solution dissolves a biomolecule modified with a modifying molecule and has a pH of more than 8.0, and the modifying molecule has a pH of the electrolyte solution. Is larger than 8.0, the passage of the nanopore is blocked by itself.

Further features related to the present disclosure will be apparent from the description of the present specification and the accompanying drawings. Also, the aspects of the present disclosure are achieved and realized by the elements and combinations of various elements, and the following detailed description and the aspects of the appended claims.
It is to be understood that the description herein is merely exemplary in nature and is not intended to limit the scope or claims of the disclosure in any way.

According to the present disclosure, generation of background noise can be suppressed and target molecules can be measured with a high SN ratio.
Problems, configurations, and effects other than those described above will be clarified by the following description of the embodiments.

It is a schematic diagram which shows the structure of the nanopore measurement system which concerns on 1st Embodiment. It is a schematic diagram which shows the quantification method of the measurement object molecule by a nanopore measurement system. It is a schematic diagram (a) which shows a molecule | numerator for measurement, and a schematic diagram (b) which shows a mode of introduction | transduction of a test substance into nanopore. It is a figure which shows the evaluation result of Experimental example 1. It is a figure which shows the evaluation result of Experimental example 2. It is a figure which shows the evaluation result of Experimental example 3. It is a schematic diagram which shows the hardware external appearance structure of the nanopore measurement system which concerns on 1st Embodiment. It is a flowchart which shows the pre-processing method of a sample. It is a schematic diagram which shows the binding | bonding method of the modification molecule | numerator which concerns on 2nd Embodiment. It is a schematic diagram which shows the binding | bonding method of the modification molecule | numerator which concerns on 2nd Embodiment. It is a schematic diagram which shows the binding | bonding method of the modification molecule | numerator concerning 3rd Embodiment. It is a schematic diagram which shows the structure of the nanopore measurement system which concerns on 4th Embodiment.

Hereinafter, an embodiment of the present disclosure will be described based on the drawings. It should be noted that although the accompanying drawings show specific embodiments according to the principles of the present disclosure, they are for understanding of the present disclosure, and are not used for limiting interpretation of the present disclosure. It is not something that can be done.

[First Embodiment]
FIG. 1 is a schematic diagram showing the configuration of the nanopore measurement system according to the first embodiment. The nanopore measurement system includes the biomolecule analysis device 1 and a computer terminal 108. The biomolecule analysis apparatus 1 includes a biomolecule analysis device 100, an ammeter 106 (measurement unit), and a power supply 107. In the present embodiment, the measurement target molecule 109 to be analyzed may be, for example, a biomolecule such as DNA or RNA having a nucleic acid as a monomer, or a polypeptide or protein having an amino acid as a monomer. In the present embodiment, as an example, an example in which the biomolecule analyzer 1 is a molecular counting device that counts the DNA to be measured will be described.

The biomolecule analysis device 100 includes a nanopore device 102 having a nanopore 101, liquid tanks 104A and 104B (a pair of liquid tanks), and electrodes 105A and 105B (a pair of electrodes). As a nanopore measurement method by the biomolecule analysis device 100, a method of applying a voltage between the electrodes 105A and 105B to electrophorese the measurement target molecule 109 and introduce it into the nanopore 101 is adopted. The method of introducing the molecule 109 to be measured into the nanopore 101 is not limited to this, and other methods may be adopted.

The nanopore device 102 includes a thin film 102A on which the nanopore 101 is formed, and thin film fixing members 102B and 102C that sandwich the thin film 102A.

The thin film 102A may be a lipid bilayer (bio-type nanopore) composed of an amphipathic molecule layer in which a protein having a pore in the center is embedded, or a thin film composed of a material that can be formed by semiconductor microfabrication technology ( It may be a solid type nanopore). Materials that can be formed by the semiconductor fine processing technology include, for example, silicon nitride (SiN), silicon oxide (SiO ₂ ), silicon oxynitride (SiON), hafnium oxide (HfO ₂ ), molybdenum disulfide (MoS ₂ ), and graphene. Is mentioned.

The thickness of the thin film 102A can be set to 1 Å to 200 nm, and can be set to 1 Å to 100 nm or 1 Å to 50 nm depending on the situation, for example, about 5 nm.

The thin film 102A may be a laminated body. If the thin film 102A and the laminate, for example, _Si 3 _N 4/2-layer structure or of SiO _2, can be a three-layer structure in the _{Si 3 N 4 / SiO 2 /} Si 3 N 4. In the case of a three-layer structure, the intermediate layer may be polysilicon. The number of layers and the material of each layer are not limited to these, and can be changed arbitrarily.

The thin film 102A can be manufactured by the following procedure, for example. First, Si ₃ N ₄ / SiO ₂ / Si ₃ N ₄ is deposited at a thickness of 12 nm / 250 nm / 100 nm on the surface of a 725 μm thick 8-inch Si wafer, and Si ₃ N ₄ is deposited at a thickness of 112 nm on the back surface. Then, Si ₃ N ₄ on the uppermost surface is subjected to 500 nm square reactive etching, and Si ₃ N ₄ on the rear surface is subjected to 1038 μm square reactive ion etching. Further, with respect to the back surface, the Si substrate exposed by the etching is further etched by TMAH (Tetramethylammonium hydroxide). During Si etching, the wafer surface is covered with a protective film (ProTEK B3 primer and ProTEK B3, manufactured by Brewer Science Co., Ltd., “ProTEK” is a registered trademark) in order to prevent etching of SiO on the surface side.

Next, after removing the protective film, the SiO layer exposed in a square of 500 nm is removed with a BHF solution (HF / NH ₄ F = 1/60, 8 min). Thereby, a partition body in which the thin film Si ₃ N ₄ having a film thickness of 12 nm is exposed is obtained. If polysilicon is selected for the sacrificial layer, etching with KOH exposes thin film 102A. At this stage, the nanopore is not provided in the thin film 102A.

The size of the nanopore 101 can be selected according to the type of the modified molecule 110 of the measurement target molecule 109 which is the analysis target, and is, for example, 0.9 nm to 100 nm, and 0.9 nm to 100 nm depending on the situation. The thickness can be set to 50 nm, and specifically, it is about 0.9 nm or more and 30 nm or less. For example, as the modified molecule 110, the nanopore diameter when streptavidin having a diameter of about 10 nm is modified on the measurement target molecule 109 can be 5 nm to 30 nm, and can be 5 nm to 15 nm depending on the situation. Specifically, it can be about 10 nm. Further, for example, when dCas9 having a diameter of about 30 nm is used as the modifying molecule 110, the nanopore diameter can be set to 40 nm or less.

The depth of the nanopore 101 can be adjusted by adjusting the thickness of the thin film 102A. Further, the shape of the cross section of the nanopore 101 on the horizontal plane is basically substantially circular, but it may be substantially elliptical or polygonal.

The liquid tanks 104A and 104B are arranged so as to sandwich the nanopore device 102, and each is filled with the electrolyte solution 103. Electrode 105A is arranged in liquid tank 104A, and electrode 105B is arranged in liquid tank 104B. The liquid tanks 104A and 104B are formed of materials, shapes and sizes that do not affect the measurement of the blocking current. The capacity of the electrolyte solution 103 is, for example, on the order of microliters or milliliters.

As the electrolyte to be dissolved in the electrolyte solution 103, for example, potassium salt such as potassium chloride (KCl), ammonium salt such as ammonium chloride (NH ₄ Cl) or ammonium sulfate ((NH ₄ ) ₂ SO ₄ ), magnesium chloride (MgCl ₂ ) Commonly used electrolytes such as magnesium salts such as magnesium sulfate (MgSO ₄ ) and magnesium sulfate can be used. As the electrolyte, lithium salt, sodium salt, rubidium salt, cesium salt or the like may be used. Of these electrolytes, one kind may be used alone, or two or more kinds may be used in combination.

The electrolyte solution 103 contains a buffer. The buffer can include, for example, Tris, EDTA, phosphate buffered saline (PBS), nonionic detergent, Tris-HCl, and the like.

As the electrolyte solution 103, a solution containing (NH ₄ ) ₂ SO ₄ , KCl, MgSO ₄ , a nonionic surfactant, Tris-HCl, or the like is generally used. In order to suppress the formation of the self-complementary strand of the molecule 109 to be measured, it is possible to mix Urea of 4 M or more, DMSO, DMF, and NaOH in the liquid tank 104B on the side where the modifying molecule 110 is not introduced. A more detailed description of the electrolyte solution 103 will be given later.

The electrodes 105A and 105B are made of a material capable of performing an electron transfer reaction (Faraday reaction) with the electrolyte in the electrolyte solution 103, and typically made of silver halide or alkali silver halide. From the viewpoint of potential stability and reliability, silver or silver chloride can be used.

The electrodes 105A and 105B may be made of a material that becomes a polarized electrode, for example, gold or platinum. In that case, in order to secure a stable ionic current, a substance capable of assisting the electron transfer reaction, such as potassium ferricyanide or potassium ferrocyanide, can be added to the electrolyte solution 103. Alternatively, a substance capable of performing an electron transfer reaction, such as ferrocene, can be immobilized on the surface of the polarized electrode.

The electrodes 105A and 105B may be entirely made of the above material, or the surface of a base material (copper, aluminum, etc.) may be coated with the above material. The shapes of the electrodes 105A and 105B are not particularly limited, but a shape having a large surface area in contact with the electrolyte solution 103 can be adopted.

The electrodes 105A and 105B are joined to the wiring, and an electric signal is sent to the measuring circuit of the ammeter 106. Although not shown in FIG. 1, connection terminals electrically connected to the electrodes 105A and 105B are provided on the outer peripheral surface of the biomolecule analysis device 100, whereby the electrodes 105A and 105B are connected to the power source 107 and It is connected to the ammeter 106.

The ammeter 106 measures an ionic current flowing between the electrodes 105A and 105B. Although not shown, the ammeter 106 has an amplifier that amplifies the current flowing between the electrodes 105A and 105B, and an analog / digital converter. The ammeter 106 is connected to the computer terminal 108, for example, by wire or wirelessly, and outputs the current value of the detected ion current from the analog / digital converter to the computer terminal 108 as a digital signal.

The computer terminal 108 is, for example, a terminal such as a personal computer, a smartphone, or a tablet, and has a data processing unit that processes various data. The data processing unit counts the molecules to be measured 109 or acquires monomer sequence information of biomolecules based on the current value of the ionic current output from the ammeter 106. The computer terminal 108 also includes a storage unit that stores the output value of the ammeter 106, data calculated by the data processing unit, and the like.

When a voltage is applied from the power supply 107 to the electrodes 105A and 105B, an electric field is generated in the vicinity of the nanopore 101, and the measurement target molecule 109 negatively charged in the electrolyte solution 103 is electrophoresed and passes through the inside of the nanopore 101. At that time, a blocking current flows. In the blockade current measurement using the biomolecule analysis device 100, the current value measured in the absence of the measurement target molecule 109 is used as a reference (pore current), and the current observed when the measurement target molecule 109 is encapsulated decreases. (Blocking of the nanopore 101 by the measurement target molecule 109) is measured, and the passing speed and state of the molecule are observed. When the measurement target molecule 109 has finished passing through the nanopore 101, the acquired current value returns to the pore current. It is possible to analyze the nanopore passage speed of the measurement target molecule 109 from this blocking time, and to analyze the characteristic of the measurement target molecule 109 from the blocking amount.

As the nanopore measurement method, it is possible to adopt the following method in addition to the method of measuring the blocking current as described above. One is a method in which, in addition to the electrodes 105A and 105B, another pair of electrodes is provided in the vicinity of the nanopore, a voltage is applied between the pair of electrodes, and a change in tunnel current generated when a biomolecule passes through is measured. is there. In addition, there is a method of providing a FET device on a nanopore membrane and measuring a signal change of a transistor acquired by the device. It is also possible to measure optical signals such as absorption, reflection and fluorescence characteristics of the light irradiated near the nanopore.

As shown in FIG. 1, the power supply 107, the ammeter 106, and the computer terminal 108 are not separate members from the biomolecule analysis device 100, but the power supply 107, the ammeter 106, and the computer terminal 108 are used for biomolecule analysis. It may be integrated with the device 100. Although not shown, the biomolecule analysis device 100 includes a sample introduction port for introducing a sample such as the measurement target molecule 109 into the electrolyte solution 103.

Next, a method of forming the nanopore 101 in the thin film 102A will be described. The nanopore 101 is formed on the thin film 102A by, for example, electron beam irradiation by a transmission electron microscope or the like, or dielectric breakdown by voltage application.

The nanopore 101 can be formed by applying a voltage, for example, by the following procedure. Before setting the thin film 102A on the biomolecule analysis device 100, the Si ₃ N ₄ thin film is made hydrophilic by Ar / O ₂ plasma (manufactured by Samco Co., Ltd.) under the conditions of 10 W, 20 sccm, 20 Pa, and 45 sec. Next, the thin film 102A is set on the biomolecule analysis device 100. After that, the liquid tanks 104A and 104B are filled with the electrolyte solution 103, the electrodes 105A and 105B are introduced into the liquid tanks 104A and 104B, and a voltage is applied.

Here, the liquid tank 104B located on the lower side is called a cis tank, and the liquid tank 104A located on the upper side is called a trans tank. The voltage Vcis applied to the electrode on the cis tank side is set to 0 V, and the voltage Vtrans is applied to the electrode on the trans tank side. The voltage Vtrans is applied as a pulse voltage by, for example, a pulse generator (41501B SMU AND Pulse Generator Expander, manufactured by Agilent Technologies). The process of applying a voltage to form the nanopore 101 is controlled by, for example, a self-made program (Excel VBA, Visual Basic for Applications).

The current value after applying the pulse voltage can be read by the ammeter 106 (4156B PRECISION SEMICONDUCTOR ANALYZER, manufactured by Agilent Technologies). The diameter of the nanopore 101 can be estimated from the ion current value. A current value condition (threshold current) is selected according to the diameter of the nanopore 101 formed before the application of the pulse voltage, and the diameter of the nanopore 101 is successively increased to obtain the target diameter.

Table 1 shows the criteria for selecting the conditions. Here, the n-th pulse voltage application time t _n (where n> 2 is an integer) is determined by the following equation.

The timing of forming the nanopore 101 may be at the time of shipment or immediately before measurement. Generally, the nanopore 101 formed by EB lithography or electron beam irradiation is formed before shipping. The nanopore 101 formed by applying a voltage may be before shipment or immediately before measurement. After forming the nanopore 101 or after forming the thin film 102A, the hydrophilicity of the nanopore 101 can be maintained by immersing it in a salt solution or the like before shipping. Further, by this, it is possible to avoid the bubbles from being bitten when the liquid is sent during the measurement.

As shown in FIG. 1, the measurement target molecule 109 is introduced into the electrolyte solution 103 together with the modifying molecule 110. The measurement target molecule 109 may be bound to the modification molecule 110 in a buffer in advance and then introduced into the electrolyte solution 103. Here, by mixing the modification molecule 110 capable of binding to the measurement target molecule 109 in an excessive amount with respect to the measurement target molecule 109, the quantitativeness of the measurement target molecule 109 can be improved. As a result, a certain number of modified molecules 110 that have not bound to the measurement target molecule 109 remain in the electrolyte solution 103.

When the measurement target molecule 109 is a nucleic acid, the measurement target molecule 109 is negatively charged, and therefore the measurement target molecule 109 is attracted to the positive electrode side by applying a voltage to the electrodes 105A and 105B, and is introduced into the nanopore 101. It In the electrolyte solution 103, in addition to the measurement target molecule 109 modified with the modification molecule 110, the non-measurement target molecule 114 and the modified molecule 110 that cannot bind to the measurement target molecule 109 are mixed.

FIG. 2 is a schematic diagram showing a method of quantifying the measurement target molecule 109 by the nanopore measurement system of the present embodiment. The biomolecule analysis device 100 includes a sample introduction port 140, a nanopore device 102, and an electrode substrate 150, and is used as a disposable chip. In the example shown in FIG. 2, the nanopore device 102 is an array device having a plurality of nanopores 101.

The biomolecule analysis device 100 is connected to the measuring instrument 170 by wiring or the like connected to the electrodes 105A and 105B, respectively. The measuring instrument 170 is equipped with an ammeter 106, a power source 107, a control circuit (not shown) and the like, and is configured to be capable of wired or wireless communication with the computer terminal 108.

In the electrolyte solution 103, measurement target molecules A to C (measurement target molecule 109), probes A to C (modification molecule 110) capable of binding to them, respectively, and non-measurement target molecule D (non-measurement target) are provided as analytes (samples). The molecule to be measured 114) is introduced from the sample introduction port 140 of the biomolecule analysis device 100 using, for example, the dispensing nozzle 160.

As shown in FIG. 2, as the modified molecule 110, different sizes (volumes) and different electrical characteristics are used according to the type and characteristics of the measurement target molecule 109, so that the measurement target molecule 109 can be adjusted. Since different blocking current amounts and signal durations are measured, it is possible to count each measurement target molecule 109. In the example shown in FIG. 2, the measurement target molecules A to C and the non-measurement target molecule D are single-stranded DNAs, but the present invention is not limited to this, and even if they are wholly or partially double-stranded. Good.

After introducing the sample into the biomolecule analysis device 100, the user drives the power supply 107 to apply a voltage between the electrode 105A and the plurality of electrodes 105B. As a result, the measurement target molecule 109 and the non-measurement target molecule 114, which are negatively charged, are introduced into the nanopore 101 due to the potential gradient formed in the vicinity of the nanopore 101. When these analytes pass through the nanopore 101, the nanopore 101 is blocked, so that the ionic current between the electrodes 105A and 105B decreases, and the analyte passage event is observed as an output signal of the ammeter 106.

In the example shown in FIG. 2, as an output signal of the ammeter 106, an example of the result of the sample passage event in the nanopores 101a to 101c is shown. One sample passage event indicates that one measurement target molecule has passed through the nanopore 101, and the current value and elapsed time of the sample passage event are the volume of the modified molecule 110 that modifies the measurement target molecule and the modified molecule. It depends on the interaction between 110 and the nanopore 101.

The ammeter 106 outputs a detection signal to the computer terminal 108 by communicating with the computer terminal 108. Based on the output of the ammeter 106, the computer terminal 108 plots the current value and the residence time of the measurement target molecule in the nanopore 101, calculates the abundance rate of each measurement target molecule, and displays the result. It is displayed on the (display section). The computer terminal 108 determines which measurement target molecule has passed through the nanopore 101 based on the current value and elapsed time of the sample passage event, and counts each measurement target molecule based on the number of sample passage events. can do.

However, when the modified molecule 110 is charged to the same degree as the measurement target molecule 109 in the electrolyte solution 103 and is introduced into the nanopore 101, the modified molecule 110 passes through the nanopore 101. As a result, there is no difference between the blockade signal derived from the modified measurement target molecule 109 and the blockade signal derived only from the modified molecule 110, or the blockade signal derived from the modified molecule 110 and the non-measurement target molecule 114. There will be no difference from the blocked signal, and it will be difficult to calculate the abundance rates of the measurement target molecule 109 and the non-measurement target molecule 114.

In order to prevent the modified molecule 110 from passing through the nanopore 101 by itself as described above, a method of using an uncharged (uncharged) modified molecule 110 such as PNA and polyethylene glycol (PEG) can be considered. However, various chemical reactions are required to bond PEG, and since PEG is a linear polymer, the measurement signal derived from the measurement target molecule 109 at the time of modification and the measurement signal at the time of non-modification are measured. It is difficult to take the difference from the measurement signal derived from the molecule 109.

For the above reason, in order to obtain a measurement signal with a higher SN ratio, it is considered that a protein having a larger molecular weight than the measurement target molecule 109 and having a clear difference in measurement signal is suitable as the modified molecule 110. .. However, since the protein has an isoelectric point, as described above, the protein itself is electrophoresed depending on the region of the solution, which hinders nanopore measurement. In order to solve such a problem, it is necessary to prepare a state in which only the modifying molecule 110 cannot pass through the nanopore 101.

The measurement target molecule 109, the modified molecule 110, and the non-measurement target molecule 114 introduced as the sample will be described. FIG. 3A is a schematic diagram showing the measurement target molecule 109 modified with the modification molecule 110. In the example shown in FIG. 3A, the measurement target molecule 109 is double-stranded, and the modified molecule 110 is the PNA 113 capable of binding to the measurement target molecule 109 and the linker 112 for binding the modified molecule 110 and the PNA 113. Have and.

As the modifying molecule 110, one having a larger volume than the measurement target molecule 109 and a size capable of passing through the nanopore 101 can be used. As a specific example of the modifying molecule 110, a polymer compound represented by a protein is preferable. In particular, avidin, streptavidin, neutravidin, nucleases such as inactivated Cas9 dCas9, polymerases, antibodies, antigens and the like can be mentioned. As the linker 112, a known linker such as PEG or biotin can be used.

As shown in FIG. 3A, the PNA 113 bound to the modified molecule 110 via the linker 112 invades the target site of the measurement target molecule 109, and partially forms a triple chain. As a result, the modified molecule 110 is bound to the measurement target molecule 109. Note that BNA or LNA may be used instead of PNA 113. PNA, BNA, and LNA have a sequence complementary to the target site of the measurement target molecule 109 and have stronger binding force than DNA, so that the PNA, BNA, and LNA replace the target site that is thermally dissociated and bind to the modified molecule 110. It becomes possible to modify. It is also possible to modify the modifying molecule 110 by binding PNA, BNA, or LNA to the double-stranded ditch.

FIG. 3B is a schematic diagram showing how a sample is introduced into the nanopore 101. As described above, since the modified molecule 110 is added in an excessive amount to the measurement target molecule 109, the measurement target molecule 109 to which the modification molecule 110 is bound, the non-measurement target molecule 114, and the modification molecule 110 are added to the electrolyte solution 103. Are mixed. As shown in FIG. 3B, the measurement target molecule 109 to which the modified molecule 110 is bound and the non-measurement target molecule 114 pass through the nanopore 101, and the modified molecule 110 alone does not pass through the nanopore 101. It is necessary to suppress the background noise due to the modifier molecule 110 alone.

As a result of earnest studies, the present inventors have found that the pH of the electrolyte solution 103 is set to be higher than 8.0, the modifier molecule 110 can be prevented from passing through the nanopore 101 by itself, and the background noise can be reduced. In other words, the modifying molecule 110 is blocked from passing through the nanopore 101 in a solution having a pH higher than 8.0. The pH of the electrolyte solution 103 can be 8.5 or higher, or 9.0 or higher depending on the situation. Further, when the modifying molecule 110 is a substance having an isoelectric point such as a protein, the pH of the electrolyte solution 103 can be set to the isoelectric point or higher.

Hereinafter, the pH of the electrolyte solution 103 and the passage of the modifying molecule 110 through the nanopore 101 will be described with reference to experimental examples.

<Experimental example 1>
In Experimental Example 1, the nanopore measuring system according to the first embodiment is used to evaluate passage of nanopores with streptavidin alone as the modifying molecule 110. Specifically, silicon nitride (silicon nitride) is used as the thin film 102A, and a 1M KCl aqueous solution having a pH adjusted to 3, 5, 7, 8.5, 11 or 13 is used as the electrolyte solution 103. Then, streptavidin was introduced into the liquid tank 104A, and a voltage of 0.1 V was applied between the electrodes 105A and 105B. The current value between the electrodes 105A and 105B was measured 0 to 60 seconds after the start of voltage application.

FIG. 4 is a diagram showing an evaluation result of Experimental Example 1, showing a state of streptavidin passing through the nanopore depending on the pH of the electrolyte solution 103. FIGS. 4 (a) to 4 (f) show changes with time of the ionic current intensity when the pH is 3, 5, 7, 8.5, 11 or 13, respectively. When streptavidin passes through the nanopore 101, the nanopore 101 is blocked, so that the current value flowing between the electrodes 105A and 105B decreases, and a passage event is observed in the output result of the ammeter 106. As shown in FIG. 4, it was found that the passage event of streptavidin was observed most when the pH of the electrolyte solution 103 was 5, and almost no passage event was observed when the pH was 8.5 to 13.

<Experimental example 2>
In Experimental Example 2, streptavidin is used as the modifying molecule 110, and the number of passing events of the nanopore 101 with streptavidin alone is evaluated. Specifically, as the electrolyte solution 103, a 1M KCl aqueous solution having a pH adjusted to 3, 5, 7.5, 9, 11, or 13 was used, streptavidin was introduced into the liquid tank 104A, and the electrodes 105A and 105B were introduced. In the meantime, a voltage of 0.1 V was applied for 1 minute. The number of appearances of the signal due to the passing event was counted, and the number per 30 seconds was calculated.

FIG. 5 is a diagram showing the evaluation results of Experimental Example 2, and shows the number of signal appearances of streptavidin every 30 seconds depending on the pH of the electrolyte solution 103. As shown in FIG. 5, it was found that the number of signal appearances was highest when the pH of the electrolyte solution 103 was 5, and the appearance of the signal due to streptavidin was suppressed when the pH was 9 to 13.

<Experimental example 3>
In Experimental Example 3, a 1 M KCl aqueous solution adjusted to pH 8.5 was used as the electrolyte solution 103, and a voltage of 0.2 V was applied between the electrodes 105A and 105B to perform nanopore measurement.

As a sample, as shown in Table 2 below, a double-stranded DNA modified with streptavidin (SA) and biotin as the modified molecule 110 and the measurement target molecule 109, respectively, was prepared. Streptavidin is modified into double-stranded DNA by the biotin-streptavidin reaction.

First, after measuring the nanopore before introducing the sample, the biotin-modified double-stranded DNA was introduced into the electrolyte solution 103, and the nanopore was measured. Then, the biotin-modified double-stranded DNA and streptavidin were bound in a buffer, introduced into the electrolyte solution 103, and nanopore measurement was performed.

FIG. 6A is a graph showing the time change of the voltage value in Experimental Example 3. As shown in FIG. 6 (a), a signal does not appear before the introduction of the sample, and a blocking signal is confirmed when biotin-modified double-stranded DNA (dsDNA) is introduced. Further, it was found that when a double-stranded DNA bound with streptavidin (SA-modified dsDNA) was introduced, a blocking signal larger than that of the double-stranded DNA alone was obtained.

FIG. 6B is a scatter diagram (left side) and a histogram (right side) showing the evaluation result of the blocking current amount in Experimental Example 3. The histogram on the right side of FIG. 6B is a count of the number of appearances for each blocking current amount. Gaussian fitting is performed on this histogram, and the amount ratio of streptavidin-unmodified double-stranded DNA (dsDNA) and streptavidin-modified double-stranded DNA (SA-modified dsDNA) is calculated from the area ratio of Gaussian distribution. You can

As shown in FIG. 6 (b), it was found that the signal derived from the streptavidin-unmodified double-stranded DNA and the signal derived from the streptavidin-modified double-stranded DNA were significantly different in the amount of blocking signal.

As described above, from the evaluation results of Experimental Examples 1 to 3, when streptavidin is used as the modifying molecule 110 and the pH of the electrolyte solution 103 is set to be higher than 8.0, the passage of streptavidin alone, which may be the background, into nanopores is suppressed. It was shown that a signal derived from unmodified double-stranded DNA can be distinguished from a signal derived from modified double-stranded DNA.

Normally, the surface of a protein is negatively charged in a solution having a pH above the isoelectric point of the protein. Further, in the nanopore measurement, a substance having a negative charge is attracted to the positive electrode and passes through the nanopore, at which time a blocking signal is confirmed.

However, despite the isoelectric point of streptavidin being 6.5 to 7.5, the blocking signal was not confirmed at pH 8.5 or higher as described above, and the signal appearance amount was the highest near pH 5. ..

The reason is that the thin film 102A in which the nanopores 101 are formed is silicon nitride, and since SiO ⁻ also exists, the surface charge of the channel through which the molecule passes is negatively charged, and the channel of the nanopore 101 is charged. It is considered that this is because many cations are easily present inside. In addition, it is considered that when an electric field is applied through the nanopore 101, cations inside the nanopore 101 are attracted to the negative electrode, and an electroosmotic flow is generated from the positive electrode toward the negative electrode. In addition, it has been calculated that the effect becomes greater as the salt concentration and pH increase (Yeh, L. et al., Analytical chemistry, 85, 7527-7534 (2013)).

Therefore, under pH conditions higher than the isoelectric point, especially in alkaline solutions, the force exerted by the electroosmotic flow outweighs the electrostatic charge of the protein itself, and nanopore passage is no longer confirmed.

On the other hand, it was confirmed that the nanopores passed under the pH condition smaller than the isoelectric point of the protein, especially on the acidic side, because it was completely unaffected by the electroosmotic flow and was induced by the electric field due to a slight negative charge. It is thought that this is because it passed through the nanopore. Under a pH condition much lower than the isoelectric point, the positive charge on the protein surface becomes dominant and the passage of nanopores is suppressed. Actually, the result of protein measurement by atomic force microscope (AFM) suggests that it may be negatively charged near the isoelectric point (Almonte, L. et al., ChemPhysChem, 15, 2768). -2773, (2014)).

For the above reasons, the amount of the measurement target molecule 109 and the ratio of the measurement target molecule 109 to the non-measurement target molecule 114 are measured while suppressing the signal derived from the modified molecule 110 alone under the specific solution conditions. It is considered possible to do so.

It was confirmed that streptavidin, even if it had different isoelectric points, showed no signal when compared to neutral conditions under alkaline conditions, especially when the pH was raised to about 11.

FIG. 7 is a schematic diagram showing a hardware appearance configuration of the nanopore measurement system according to the present embodiment. In the example shown in FIG. 7, the computer terminal 108 is a notebook computer, and the biomolecule analysis device 1 is provided as a thin device. As shown in FIG. 7B, the biomolecule analysis apparatus 1 is configured by integrating the biomolecule analysis device 100, the control circuit unit 703, and the simple display 705.

Although not shown, the control circuit unit 703 includes the above-described ammeter 106 and power supply 107, a control circuit, and a communication interface. The control circuit is a circuit for controlling the operation of each component of the control circuit unit 703. A voltage is applied between the electrodes 105A and 105B by the power supply 107, and an ionic current (blocking current) between the electrodes 105A and 105B is measured by the ammeter 106. The communication interface is configured to be able to send the output signal of the ammeter 106 to the computer terminal 108.

The simple display 705 is configured to be able to display various data such as an output signal received from the ammeter 106, a voltage value between the electrodes 105A and 105B, and a measurement time.

As shown in FIG. 7C, the biomolecule analysis device 100 is configured to be separable from the control circuit unit 703 and the simple display 705 and is disposable. The biomolecule analysis device 100 includes the nanopore device 102 and the sample introduction port 140 described above, a flow path for enclosing a solution, and a flow cell forming the liquid tanks 104A and 104B. The sample including the measurement target molecule 109, the modified molecule 110, and the like is introduced into the liquid tank 104A from the sample introduction port 140 after performing a predetermined pretreatment. At this time, the electrolyte solution 103 having a pH higher than 8.0 coexists in the liquid tank 104A. The system for pretreatment of the sample may be integrated with the biomolecule analysis device 100. In this case, the biomolecule analysis device 100 includes a stirring system for pretreatment, a liquid feeding system, a temperature control system, and the like. It may be installed.

FIG. 8 is a flowchart showing a sample pretreatment method. First, in step S10, a user uses an extraction kit or the like to extract DNA (biomolecule) from, for example, plant cells. Then, in step S21, the user amplifies a specific target region of DNA using the primer with a bead binding site. In step S30, the magnetic beads are bound to the bead binding sites of the primer to recover a specific target region (measurement target molecule) of DNA. In step S40, the measurement target molecule to which the magnetic beads are bound is introduced into the biomolecule analysis apparatus 1 as a sample, and nanopore measurement is performed.

As another pretreatment method, instead of step S21 described above, in step S22, a primer for detecting a specific site may be bound to the extracted DNA to amplify the site to be the molecule to be measured. Then, in step S30, the amplified DNA is collected by the magnetic beads. Next, in step S31, the magnetic beads are removed, and in step S32, the measurement probe having the modifying molecule is bound. In step S40, the measurement target molecule bound to the measurement probe is introduced as a sample into the biomolecule analysis apparatus 1 to perform nanopore measurement.

As yet another pretreatment method, instead of step S21, the extracted DNA may be fragmented to obtain a plurality of fragments containing the molecule to be measured, as step S23. Then, steps S30 to S32 and S40 are performed in the same manner as above.

The nanopore device for analyzing biomolecules according to the present embodiment includes the above-mentioned configuration as an element. The nanopore device can be provided together with instructions describing use procedures, usage amounts, and the like. The electrolyte solution may be provided in a ready-to-use state or may be provided in a state where only the biomolecule to be measured is not mixed. Such forms and preparations will be understood by those of ordinary skill in the art. Similarly, regarding the nanopore device, the nanopore may be provided in a state in which the nanopore is formed in a ready-to-use state, or may be provided in a state in which the nanopore is formed at a destination.

As described above, in the biomolecule analysis apparatus 1 of the present embodiment, the pH of the electrolyte solution 103 is higher than 8.0, and the modifying molecule 110 alone is not introduced into the nanopore 101. In addition, the blockade signal derived from the measurement target molecule 109 to which the modified molecule 110 is bound becomes a larger signal than the blockade signal derived from the molecule to which the modified molecule 110 is not bound. Thereby, the generation of background noise can be suppressed, and the blockade signal derived from the measurement target molecule 109 to which the modified molecule 110 is bound and the blockade signal derived from the non-measurement target molecule 114 can be obtained with a high SN ratio. . Therefore, it becomes easy to identify the measurement target molecule 109 to which the modified molecule 110 is bound, and the measurement accuracy can be improved.

The biomolecule analyzer of the present embodiment is useful in the fields of, for example, analysis of biomolecules composed of nucleic acids, and tests, diagnoses, treatments, drug discovery, basic research, etc. that use the analysis information.

[Second Embodiment]
In the above-described first embodiment, an example in which the modified molecule 110 is modified into the measurement target molecule 109 by inserting the PNA 113 to which the modified molecule 110 is bound into the double strand has been described, but in the second embodiment, Is different from the first embodiment in the modified portion of the modified molecule 110. The biomolecule analysis device and the analysis method according to the second embodiment are the same as those in the first embodiment, and thus the description thereof will be omitted.

FIG. 9 is a schematic diagram showing a method of binding the modified molecule 110 according to the second embodiment. In the example shown in FIG. 9, the modified molecule 110 is connected via a linker 112 to a primer 115 capable of binding to a specific target region of the measurement target molecule 109. The primer 115 is used when amplifying the target region. By directly binding such a primer 115 to the double-stranded molecule to be measured 109, the modifying molecule 110 can be modified only on the target site.

FIG. 10 is a schematic diagram showing another binding method of the modified molecule 110 according to the second embodiment. In the example shown in FIG. 10, the modified molecule 110 is bound by the Nick enzymatic method. That is, the modifying molecule 110 is bound to the PNA 113 (probe) complementary to the specific sequence 116 in advance, and the specific sequence 116 of the double-stranded DNA (measurement target molecule 109) is cleaved using a nicking endonuclease. Then, the measurement target molecule 109 is modified by binding the PNA 113 to the dissociated single-stranded sequence 116. The cut portion of the array 116 is repaired to form the array 117.

As described above, according to this embodiment, the modified molecule 110 can be modified on the measurement target molecule 109 by an optimum means according to the type and characteristics of the measurement target molecule 109, the purpose of measurement, and the like.

[Third Embodiment]
When the measurement target molecule 109 is recovered by fragmentation of long-chain DNA, a plurality of fragments coexist. In such a case, as a method of separating the signal derived from the measurement target molecule 109 and the signal derived from the non-measurement target molecule 114, one is to increase the target region only by amplification so as to reduce the influence of other fragments. It may be suppressed. In addition, as another means, it is possible to separate the signal derived from the measurement target molecule 109 from the signal derived from the non-measurement target molecule 114 by performing different modification on the fragmented sample according to each feature. it can.

In the present embodiment, an example of measuring the abundance rate of gene recombination sequences (GMO sequence) will be described. FIG. 11 is a schematic diagram showing the method of binding the modified molecule 110 according to the third embodiment, and FIG. 11 (a) shows the procedure for measuring the abundance ratio of the gene recombination target sequence. As shown in FIG. 11A, first, the genome (sample) is fragmented, and the measurement target molecule 109 in which the gene recombination sequence is inserted, the measurement target molecule 109 in which the gene recombination sequence is not inserted, The measurement target molecule 114 is obtained. Then, the modified molecule 110 is modified as follows with respect to the measurement target molecule 109 in which the gene recombination sequence has been inserted and the measurement target molecule 109 in which the gene recombination sequence has not been inserted, and nanopore measurement is performed. The abundance ratio of each sequence is calculated.

FIG. 11B is a schematic diagram showing an example of a modification method of the modification molecule 110 with respect to the fragmented sequence. As shown in the left and center views of FIG. 11B, the modifying molecule 110a is modified at a site common to the measurement target molecule 109 having the target site for insertion of the gene recombination sequence. Further, a modifying molecule 110b capable of binding to the gene recombination sequence is attached to the gene recombination sequence. As a result, one or more modified molecules are bound to the measurement target molecule 109 having the site of insertion of the gene recombination sequence, so that only signals derived from those to which the modified molecules are bound are counted. do it. Since the signal values of two modified molecules bound to each other and one modified molecule bound to each other differ from each other, the signals derived from them should be distinguished from each other. You can Thereby, the abundance ratio of the measurement target molecule 109 in which the gene recombination sequence is inserted and the measurement target molecule 109 in which the gene recombination sequence is not inserted can be easily calculated.

[Fourth Embodiment]
FIG. 12 is a schematic diagram showing the configuration of the nanopore measurement system according to the fourth embodiment. The nanopore measurement system of this embodiment includes a biomolecule analysis device 2 and a computer terminal 108. The biomolecule analysis device 2 of the present embodiment is the first embodiment in that the biomolecule analysis device 200 includes a nanopore device 102 having a plurality of nanopores 101, and a liquid tank 104B is divided into a plurality of liquid tanks. Different from

The nanopore device 102 is composed of a thin film 102A on which a plurality of nanopores 101 are formed, and thin film fixing members 102B and 102C that sandwich the thin film 102A. The nanopore 101 may be formed at any position on the thin film 102A. The interval at which the plurality of thin films 102A are arranged can be set to 0.1 μm to 10 μm depending on the electrodes used and the capacity of the electrical measurement system, and can be set to 0.5 μm to 4 μm depending on the situation.

The thin film fixing member 102B and the thin film 102A form a part of the structure of the liquid tank 104A. Further, the thin film 102A and the thin film fixing member 102C form a part of the structure of the liquid tank 104B. The thin film fixing member 102C has four spaces separated by three partition walls, and these spaces are respectively used as the liquid tank 104B. The liquid tank 104A is used as a common liquid tank for the four liquid tanks 104B located on the lower side. The number of liquid tanks 104B is not limited to four and can be changed arbitrarily.

The electrode substrate 150 includes a plurality of electrodes 105B that are in contact with the electrolyte solution 103 in each liquid tank 104B. As described above, each liquid tank 104B is provided with the single nanopore 101 and the electrode 105B, and the liquid tanks 104B are insulated from each other by the partition wall of the thin film fixing member 102C. Therefore, the current flowing through each nanopore 101 can be measured independently.

Since the biomolecule analysis method in this embodiment is the same as that in the first embodiment, description thereof will be omitted.

As described above, according to the biomolecule analysis apparatus 2 of the present embodiment, the plurality of nanopores 101 is provided, so that nanopore measurement can be performed efficiently.

[Modification]
The present disclosure is not limited to the above-described embodiments and includes various modifications. For example, the above-described embodiment has been described in detail for the purpose of easily understanding the present disclosure, and does not necessarily have to include all the configurations described. Further, a part of one embodiment can be replaced with the configuration of another embodiment. Further, the configuration of another embodiment can be added to the configuration of one embodiment. Further, a part of the configuration of each embodiment may be added, deleted, or replaced with a part of the configuration of another embodiment.

1, 2 ... Biomolecule analyzer 101 ... Nanopore 102 ... Nanopore device 103 ... Electrolyte solution 104 ... Liquid tank 105 ... Electrode 106 ... Ammeter 107 ... Power supply 108 ... Computer terminal 109 ... Measurement molecule 110 ... Modification molecule 112 ... Linker 113 … PNA
114 ... Non-measurement target molecule 115 ... Primer 116, 117 ... Array 140 ... Sample introduction port 150 ... Electrode substrate 160 ... Dispensing nozzle 703 ... Control circuit unit 705 ... Simple display

Claims (15)

1. A thin film having nanopores,
   A pair of liquid tanks arranged so as to sandwich the thin film and containing an electrolyte solution,
   A pair of electrodes arranged in each of the pair of liquid tanks,
   A measuring unit connected to the pair of electrodes,
   In the electrolyte solution, a biomolecule modified with a modifying molecule is dissolved, and the pH is higher than 8.0.
   The biomolecule analysis device, wherein the modifying molecule alone blocks passage of the nanopore when the pH of the electrolyte solution is higher than 8.0.
2. The biomolecule analyzer according to claim 1,
   The biomolecule analysis device, wherein the electrolyte solution has a pH of 8.5 or more.
3. The biomolecule analyzer according to claim 1,
   The biomolecule analysis device, wherein the electrolyte solution has a pH of 9 or more.
4. The biomolecule analyzer according to claim 1,
   A biomolecule analyzer, wherein the modified molecule is a protein.
5. The biomolecule analyzer according to claim 1,
   The biomolecule analyzer, wherein the modified molecule is streptavidin.
6. The biomolecule analyzer according to claim 1,
   The biomolecule is double-stranded,
   A biomolecule analyzer, wherein PNA, BNA or LNA to which the modifying molecule is bound is inserted at a specific position of the double chain.
7. The biomolecule analyzer according to claim 1,
   The biomolecule is a double-stranded chain having a primer portion,
   The biomolecule analyzer, wherein the modifying molecule binds to one or more places of the primer part.
8. The biomolecule analyzer according to claim 1,
   The biomolecule is double-stranded,
   A biomolecule analyzer, wherein PNA, BNA, or LNA to which the modifying molecule is bound is bound to the cleaved portion of the double strand.
9. The biomolecule analyzer according to claim 1,
   The biomolecule analysis device, wherein the diameter of the nanopore is 5 nm to 20 nm.
10. The biomolecule analyzer according to claim 1,
    A biomolecule analyzer, wherein the thin film has a thickness of 10 nm to 30 nm.
11. The biomolecule analyzer according to claim 1,
    A biomolecule analyzer, wherein the thin film is silicon nitride.
12. The biomolecule analyzer according to claim 1,
    The biomolecule analysis device, wherein the measurement unit measures either a blocking current, a tunnel current or a transistor current of the nanopore.
13. A thin film having nanopores,
    A pair of liquid tanks arranged so as to sandwich the thin film and containing an electrolyte solution,
    A pair of electrodes arranged in each of the pair of liquid tanks,
    A step of preparing a biomolecule analysis device comprising a measurement unit connected to the pair of electrodes,
    Adjusting the pH of the electrolyte solution to be greater than 8.0,
    Introducing a modifying molecule and a biomolecule into the electrolyte solution,
    The biomolecule analysis method, wherein the modifying molecule alone blocks passage of the nanopore when the pH of the electrolyte solution is higher than 8.0.
14. The biomolecule analysis method according to claim 13,
    Extracting the biomolecule,
    Amplifying a region of interest in the biomolecule with a primer having a bead binding site,
    Further comprising collecting the region of interest with beads,
    In the step of introducing the modification molecule and the biomolecule into the electrolyte solution, the modification molecule and the target region bound to the beads are introduced into the electrolyte solution.
15. The biomolecule analysis method according to claim 13,
    Extracting the biomolecule,
    A step of amplifying a target region in the biomolecule by a specific site detection primer, or a step of fragmenting the biomolecule to obtain a target region in the biomolecule, and
    Binding the region of interest to magnetic beads and collecting the magnetic beads with a magnet;
    Further removing the region of interest from the magnetic beads,
    In the step of introducing the modifying molecule and the biomolecule into the electrolyte solution, the target region and the modifying molecule are introduced into either one of the pair of liquid tanks.

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