JP2015206737A - analyzer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve problems that, for example, in a conventional method, for detecting a trace quantity of a nucleic acid with high sensitivity by amplifying the nucleic acid included in a sample in a manner specific to a base sequence, fluorescence dye is used for a flag of the nucleic acid, and change of fluorescence intensity is traced with time for analyzing, so that it is necessary to amplify the nucleic acid until detecting the fluorescence, and the change of fluorescence intensity in which the change of fluorescence intensity is in secular change has to be traced, and consequently, for amplification of the nucleic acid, 1-2 hours are required, and through-put is extremely bad.SOLUTION: A nucleic acid analyzer uses a nanopore-based analysis in detection of the nucleic acid.

Description

  The present invention relates to an analyzer for analyzing a biological substance, for example, an analyzer for analyzing a nucleic acid.

  A nucleic acid amplification method has been used as a method for analyzing blood, urine and the like. The nucleic acid amplification test apparatus includes a rotatable carousel, a plurality of reaction vessels mounted along the circumferential direction of the carousel, a light source for irradiating the reaction vessel with excitation light, and the excitation light in the reaction vessel. One having a detector for detecting fluorescence emitted from the reaction solution and a temperature adjusting mechanism for setting the temperature of the reaction solution in the reaction vessel to a predetermined temperature is known (see Patent Document 1). For example, when a genetic test based on the PCR (Polymerase Chain Reaction) method is performed using this test apparatus, the temperature in the casing is adjusted by the above-described heat source or the like, and the reaction in the reaction vessel disposed in the carousel is performed. The reaction solution is subjected to PCR treatment between a plurality of temperatures (for example, 95 ° C. and 59 ° C.), and fluorescence detection is performed by the above-described detector.

  On the other hand, development of a nanopore analysis method for analyzing a polymer such as DNA or protein using nanopores called nanopores has been developed (Patent Document 2). Technologies for detecting macromolecules by nanopore analysis include a blocking current method, a tunneling current method, and a capacitance method.

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

  The tunnel current method means that when a polymer passes through a nanopore, the tunnel current flows through a small gap between the tunnel current electrode and the polymer near the nanopore, and the presence of the polymer is detected. It is a method of analyzing ingredients.

  Capacitance method means that when the polymer passes through the nanopore, the nanopore is partially blocked, so the capacitor of the membrane having the nanopore changes, and the presence and components of the polymer are analyzed by detecting it. Is the method.

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

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

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

JP 2010-151665 A Special table 2011-527191 gazette

  Conventionally, in order to detect a very small amount of nucleic acid with high sensitivity by amplifying nucleic acid contained in a sample in a base sequence-specific manner, a fluorescent dye is used for nucleic acid labeling, and the change in fluorescence intensity is followed over time. Since it was necessary to follow the fluorescence intensity change that has changed over time, the nucleic acid amplification took 1-2 hours. The throughput was very bad.

  On the other hand, in order to measure the concentration of nucleic acid in nanopore analysis, it was measured by the duration and degree of current interruption. However, the passage of nucleic acids through the pores is not regular, and even if the duration and degree of current interruption at a certain time are measured, they do not always accurately reflect the information on the nucleic acid concentration.

  Therefore, it is necessary to grasp the nucleic acid concentration by allowing almost all of the amplified nucleic acid sample to pass through the pores. However, if the conventional nanopore detection mechanism is used as it is, if the concentration of the amplified nucleic acid sample is too high, the passage through the pores takes a long time.

  An object of the present invention is to provide a nuclear inspection analyzer and a nucleic acid analysis method for minimizing nucleic acid amplification time by PCR and detecting amplified nucleic acid with high sensitivity by a nanopore analysis method.

The nucleic acid analyzer of the present invention comprises:
A temperature control unit for controlling the temperature of a nucleic acid sample, a nanopore detection unit that has a nanometer-size pore and a pair of electrodes, detects a nucleic acid sample that has passed through the pore by electrophoresis, and a nanopore detection from the temperature control unit A transport unit for transporting the nucleic acid sample to the unit,
The nucleic acid sample amplified by the temperature control unit is counted, and the concentration of the nucleic acid sample is measured.

  According to the present invention, it is possible to achieve higher throughput and higher specimen processing efficiency than the conventional fluorescence detection method. Also, as a nanopore analysis method, analysis can be performed with higher sensitivity than before, and the DNA concentration can be calculated.

It is a figure which shows the outline | summary of the nucleic acid analyzer of this invention. The figure explaining the test | inspection process performed at the time of sample sample introduction. The figure explaining the structural example 1 of the nanopore analysis mechanism of a nucleic acid test | inspection apparatus. The figure explaining the detection process of a nanopore analysis mechanism in the structural example 1. FIG. The figure explaining the structural example 2 of the nanopore analysis mechanism of a nucleic acid test | inspection apparatus. The figure explaining the detection process of a nanopore analysis mechanism in the structural example 2. FIG. Conceptual diagram of measurement of DNA sample without primer by nanopore analysis mechanism Conceptual diagram of measurement of DNA sample with primer by nanopore analysis mechanism

  FIG. 1 shows an outline of the nucleic acid analyzer of this example. The nucleic acid analyzer of the present embodiment is composed of three main components: an extraction unit 1, a preparation unit 2, and a measurement unit 3. These three configurations are connected by the transport mechanism 4. FIG. 2 shows a diagram for explaining a test process executed at the time of introducing the specimen sample.

  The extraction unit 1 may have any configuration as long as the sample can be introduced from the sample sample introduction unit 5 and the sample can be dissolved to extract the nucleic acid in the sample. The specimen sample is, for example, before extraction of nucleic acids such as serum, plasma, urine, stool, sputum and the like. The specimen sample introduction unit 5 is a mechanism that can construct and introduce a container (for example, a blood collection tube) in which a specimen sample is sealed. In the nucleic acid extraction mechanism 141, for example, a specimen sample dissolved in a column packed with a nucleic acid binding carrier is passed through, and nucleic acid is extracted (S1). As the liquid passing method, there are a method using a centrifuge and a method of pressurizing with a syringe. Nucleic acid binding carriers include magnetic particles coated with silica and a method of collecting the magnetic particles with a magnet. The nucleic acid sample from which the nucleic acid is extracted from the specimen sample in the extraction unit 1 is transported to the preparation unit 2 by the transport mechanism 4.

  There are two types of routes through which the nucleic acid sample is introduced into the preparation unit 2. There are a route through which the nucleic acid sample is introduced from the extraction unit 1 by the transport mechanism 4 and a route through which the nucleic acid sample introduction unit 6 introduces the route. The preparation method of the reaction solution in the preparation unit 2 differs depending on the nucleic acid amplification method in the measurement unit 3. Nucleic acid amplification methods include PCR (Polymerase Chain Reaction), TMA (Transcription Mediated Amplification), NASBA (Nucleic Acid Sequence-Based Amplification), LCR (Ligase Chain Reaction), etc. The invention proposed in the specification is not limited. In this embodiment, the nucleic acid amplification method in the measurement unit 3 will be described using the PCR method as an example. The reaction solution preparation mechanism 15 prepares a PCR reaction solution (S2). The prepared reaction liquid is transported to the measuring unit 3 by the transport mechanism 4. The reaction solution preparation mechanism 15 includes a reaction container preparation mechanism and a consumables erection mechanism (not shown).

The measurement unit 3 mainly includes an amplification mechanism 7 and a nanopore detection mechanism 8. The amplification mechanism 7 performs nucleic acid amplification (S4). When the nucleic acid amplification is PCR, the number of PCR cycles may be the number of cycles in which one copy of nucleic acid molecule can be detected. More specifically, one copy may be cycled after amplification so that there is a statistically significant number in the largest chamber divided into a plurality of nanopore detection mechanisms described later, for example, statistically When a significant number is set as X copy and the PCR amplification efficiency is 100%, the necessary number of cycles n is expressed by the following equation.
(PCR amplification volume / maximum amplification volume in the chamber) x X = 2 ⁿ
According to this equation, if the PCR amplification volume is 50uL, the amplification volume in the maximum chamber is 5uL, and X is set to 30 copies, n = 8.23 and 1 copy in the amplification liquid with the number of cycles of 10 cycles or less. Can be detected quantitatively. If one copy can be detected qualitatively, X can be set to 5 copies from the Poisson distribution. In this case, n can be detected at 5.645. Thereby, measurement time can be shortened compared with the real-time PCR measurement which conventionally required 40 cycles or more. Furthermore, by suppressing the number of cycles to a minimum, excessive amplification can be suppressed and the nanopore detection time described later can be shortened.

  The amplified solution amplified by PCR is carried to the nanopore detection mechanism 8 (S4), and the nanopore detection mechanism 8 detects the amplified nucleic acid (S5). Further, the nanopore detection mechanism may be configured to have a purification channel for purifying a nucleic acid sample from an amplification solution (not shown). Thereafter, the measurement result is displayed on a display unit (not shown) (S6).

  The extraction unit 1 includes a dispensing arm 9 and a dispensing unit 11. The preparation unit 2 includes a dispensing arm 10 and a dispensing unit 12. The dispensing unit 11 moves along the dispensing arm 9 and the dispensing unit 12 moves along the dispensing arm 10. In addition, the dispensing arm 9 and the dispensing arm 10 move along the dispensing arm transport mechanism 13.

  The nanopore detection mechanism 8 of the measurement unit 3 detects the DNA concentration in the PCR-amplified solution.

  In Patent Document 2, “the frequency of current fluctuations determines the concentration of nucleotides bound in the pore. The nature of the nucleotide depends on its characteristic current signature, particularly the duration and extent of current interruption. Will be found ". However, as a result of intensive studies by the present inventors, it was found that the nucleic acid sample passed through the pores was random. If the passage is random, even if the duration and degree of current interruption per unit time at a certain time is measured, it does not necessarily accurately reflect the nucleic acid concentration information.

  Therefore, it is necessary to grasp the nucleic acid concentration by allowing almost all of the amplified nucleic acid sample to pass through the pores. However, if the conventional nanopore detection mechanism is used as it is, if the concentration of the amplified nucleic acid sample is too high, the passage through the pores takes a long time, so even if detection with nanopore technology is used, the inspection speed It cannot be speeded up.

  Therefore, in this embodiment, a section smaller than the space in the chamber is created above the pores, and an electrode is further provided in the vicinity of the section. With this configuration, even if the concentration of the amplified nucleic acid sample is too high, since the size of the compartment is smaller than the original chamber, almost all of the amplified nucleic acid sample present in the original chamber is pored. Compared to the time required to pass through, the time required to pass almost all of the amplified nucleic acid sample present in the newly provided section through the pore is short. The nucleic acid concentration is estimated from the total number of times that the amplified nucleic acid sample existing in the newly provided section passes through the pores.

  On the other hand, when the concentration of the amplified nucleic acid sample is too low, when the detection is performed in a newly provided section, the nucleic acid sample is not present in the section or the number of nucleic acid samples present in the section is extremely small. When the amount is small, there is a possibility that no nucleic acid sample passes. In these cases, the nucleic acid concentration can be estimated by using the original larger chamber.

  Hereinafter, embodiments of a nanopore analysis mechanism of a specific nucleic acid analyzer will be described with reference to the drawings.

  FIG. 3 shows one configuration of the nanopore detection mechanism. The nanopore detection mechanism is divided into regions of the first chamber 204 and the second chamber 205 across the substrate 210. The substrate 210 includes a base 202 on the second chamber 205 side, a film 203, and a coating layer (not shown) on the first chamber 204 side. Regarding the materials, the base 202 is silicon, the film 203 is silicon nitride, Silicon oxide, silicon carbide, or the coating layer is made of silicon oxide.

  The membrane 203 is provided with nanometer-size pores 201. The size of the pore 201 is desirably 1 nm to 100 nm. The method of creating the pores 201 is implemented by a method using Focused Ion Beam, a method using an electron beam, or the like. The film 203 may be silicon oxide. The coating layer has functions of insulation, strength increase, and hydrophilicity addition, and the function can be substituted by the film 203 itself.

  When the side where the PCR amplification solution is transported is upstream, the first electrode 207 is disposed upstream of the first chamber 204 and the second electrode 206 is disposed downstream of the second chamber 205. A small measurement section is provided in the vicinity of the pore 201 in the first chamber 204. The measurement section is formed by a section forming member 209. A third electrode 208 is disposed on the partition forming member 209, and the third electrode 208 is made of, for example, a cylindrical metal (metal pipe). Metal pipes are composed of silver / silver chloride, which is commonly used in the field of electrochemistry. Or you may be comprised with stainless steel, platinum, and gold | metal | money. A part of the metal pipe may be in direct contact with the ionic solution and energized. Therefore, it is desirable that the inner surface of the pipe is coated with fluorinated ethylene or the like so as not to adsorb organic substances such as a polymer.

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

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

  In the nanopore detection mechanism, the nucleic acid concentration is analyzed by detecting a physical change when the nucleic acid in the PCR amplification solution passes through the pores. FIG. 4 shows a flow of the nanopore detection mechanism. As a specific flow description, a case where the nucleic acid in the PCR amplification solution is DNA will be described. The PCR amplification solution is introduced into the nanopore detection mechanism by a flow path or dispensing to adjust the PCR amplification solution (S1). Next, the adjusted PCR amplification solution is introduced into the first chamber 204 of the nanopore detection mechanism. Next, a voltage is applied to the electrodes 206, 207, and 208 (S2).

  At this time, a voltage is applied so that the first electrode 207 and the third electrode 208 on the first chamber 204 side are negative, and the second electrode 206 on the second chamber 205 side is positive. It is preferable that the voltage of the third electrode (V3) ≦ the voltage of the first electrode (V1) <the voltage of the second electrode (V2). More preferably, V3 <V1 <V2. Due to this potential difference, ions in the solution and DNA in the PCR amplification solution move. Since DNA is negatively charged, it is attracted to the positive side. Therefore, by setting the voltage applied to the electrodes 208, 207, and 206 to V3 <V1 <V2, DNA existing in the measurement section is attracted to the second electrode 206 existing on the second chamber 205 side, and is finely divided. Pass through hole 201. At this time, the openings of the pores 201 are closed and the opening area is reduced, so that it is difficult for other ions in the liquid to flow. The difficulty of ion flow is detected as a decrease in current value (see FIG. 7b. FIG. 7a is a current waveform when the DNA to be detected does not pass through). Therefore, the total number of passes through which the DNA has passed through the pore can be found from the number of times the current value has decreased. Therefore, when the passage of DNA present in the measurement section to the pore 201 is detected and the passage is confirmed (S3), it is determined that there is a detection, and the DNA concentration is estimated from the total number of passages (S5). When the DNA concentration is low and there is almost no DNA in the measurement section, that is, when the detection of DNA cannot be confirmed within a predetermined time, the voltage of the third electrode 208 is turned off, and the first electrode 207 and the second electrode A voltage is applied between the two electrodes 206 (S4). As a result, the DNA in the first chamber 204 moves to the second chamber 205 through the pore 201. At this time, similarly, when the passage of DNA existing in the chamber 1 to the pore is detected (S6), it is determined that there is a detection, and the DNA concentration is estimated from the total number of passages to the pore 201 (S5). . If not detected (S7), the detection limit is redone from the adjustment of the PCR amplification solution (S1). With this configuration, the throughput is significantly increased as compared with the conventional fluorescence detection method. In addition, the detection time can be shortened as compared with the analysis method in which all the DNA in the first chamber 204 is always passed through the pore 201.

  As a variation of the present embodiment, as shown in FIG. 5, a plurality of measurement sections may be provided in stages. The nanopore detection mechanism shown in FIG. 6 includes a measurement section that gradually increases as the distance from the pore 301 increases in the first chamber 304. A first electrode 307 is provided in the first chamber 304, a second electrode 306 is provided in the second chamber 305, and the substrate 314 is constituted by the base 302, the film 303, and the coating film. The nanopore detection mechanism shown in FIG.

  Furthermore, partition forming members 311, 312, 313 are provided, and a third electrode 308, a fourth electrode 309, and a fifth electrode 310 are respectively disposed.

  Next, an operation flow using this nanopore detection mechanism will be described with reference to FIG. The PCR amplification solution is introduced into the nanopore detection mechanism by a flow path or dispensing to adjust the PCR amplification solution (S1). Next, the adjusted PCR amplification solution is introduced into the first chamber 304 of the nanopore detection mechanism. Next, a voltage is applied to the electrodes 306, 308, and 309. At this time, a voltage is applied so that the fourth electrode 307 and the third electrode 308 on the first chamber 304 side are negative, and the second electrode 306 on the second chamber 305 side is positive. It is preferable that the voltage of the third electrode (V3) ≦ the voltage of the fourth electrode (V4) <the voltage of the second electrode (V2). More preferably, V3 <V4 <V2.

  Due to this potential difference, ions in the solution and DNA in the PCR amplification solution move. Since DNA is negatively charged, it is attracted to the positive side. Therefore, by setting the voltage applied to the electrodes 306, 308, and 309 to V3 <V4 <V2, the DNA present in the measurement section formed by the section forming member 311 is the second electrode 306 present on the second chamber 305 side. And pass through the pore 301. At this time, the opening of the pore 301 is closed and the opening area is reduced, so that it is difficult for other ions in the liquid to flow. The difficulty of ion flow is detected as a decrease in current value. Therefore, the total number of passes through which the DNA has passed through the pore can be found from the number of times the current value has decreased. Therefore, when the passage of DNA present in the measurement section to the pore 301 is detected and passage is confirmed (S3), it is determined that there is a detection, and the DNA concentration is estimated from the total number of passages (S5).

  If the DNA concentration is low and there is almost no DNA in the measurement section formed by the section forming member 311, the third electrode 308 is turned off, and the fifth electrode 310 is turned on instead (S 4). At this time, the voltage of the fourth electrode 309 (V4) ≦ the voltage of the fifth electrode 301 (V5) <the voltage of the second electrode 306 (V2). More preferably, V4 <V5 <V2. Thus, DNA passing through the pore 301 is detected from the measurement section formed by the section forming member 312 (S6). If the DNA concentration is low and there is almost no DNA in the measurement section formed by the section forming member 312, the fourth electrode 308 is turned off and the first electrode 307 is turned on instead (S 7). At this time, the voltage of the fifth electrode 310 (V5) ≦ the voltage of the first electrode 307 (V1) <the voltage of the second electrode 306 (V2). More preferably, V5 <V1 <V2. Thus, DNA passing through the pore 301 is detected from the measurement section formed by the section forming member 313 (S8).

  Further, when the DNA concentration is low and there is almost no DNA in the measurement section formed by the section forming member 313, the fifth electrode 310 is turned off (S9). Thereby, the passage of DNA to the pore 301 is detected (S10). If this is not detected (S11), the detection limit is redone from the adjustment of the PCR amplification solution (S1).

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

201 pores
202 base
203 membrane
204 First chamber
205 Second chamber
206 Second electrode
207 first electrode
208 Third electrode
209 Compartment forming member
210 Board

Claims (6)

A temperature control unit for controlling the temperature of a nucleic acid sample, a nanopore detection unit that has a nanometer-size pore and a pair of electrodes, detects a nucleic acid sample that has passed through the pore by electrophoresis, and a nanopore detection from the temperature control unit A transport unit for transporting the nucleic acid sample to the unit,
A nucleic acid analyzer characterized by counting the number of times a nucleic acid sample amplified by a temperature control section has passed through a pore and measuring the concentration of the nucleic acid sample. In claim 1,
The nanopore detection unit includes a chamber and a partition forming member provided on one side of the substrate,
The pair of electrodes includes a first electrode provided in one region partitioned by the substrate in the chamber and a second electrode provided in the other region, and the first electrode is provided on the partition forming member. 3 electrodes are provided,
The substrate is provided with the pores, and the partition forming member is provided to surround the pores, and a nucleic acid sample is passed through the pores by applying a voltage to a region in the chamber. A nanopore detector for analyzing a nucleic acid sample,
Nucleic acid analyzer. The nucleic acid analyzer according to claim 2,
A nucleic acid analyzer comprising: a partition forming member provided with another partition forming member that forms a partition larger than the partition forming member; and a fourth electrode provided on the separate partition forming member.   The nucleic acid sample is temperature-controlled and amplified, the amplified nucleic acid sample is passed through a nanometer-size pore by electrophoresis, the number of passes through the pore is counted, and the concentration of the nucleic acid sample is measured. Nucleic acid analysis method. The nucleic acid analysis method according to claim 4,
A nucleic acid analysis method comprising passing a pore through a nucleic acid sample in a part of a compartment upstream of the pore. The nucleic acid analysis method according to claim 5,
A nucleic acid analysis method characterized by allowing a nucleic acid sample in an entire section upstream of a pore to pass through a pore when the passage of the nucleic acid sample cannot be detected within a predetermined time.