WO2024004917A1

WO2024004917A1 - Detection system, cartridge, detection method, method for producing nucleic acid probe, and method for producing biosensor

Info

Publication number: WO2024004917A1
Application number: PCT/JP2023/023497
Authority: WO
Inventors: 洋和松田; 康貴前田; 浩康田中
Original assignee: 京セラ株式会社
Priority date: 2022-06-28
Filing date: 2023-06-26
Publication date: 2024-01-04

Abstract

A nucleic acid that has been amplified in a reaction solution is detected easily and at good sensitivity. The detection system comprises a reaction unit, having an amplification unit that subjects the nucleic acid to an amplification reaction in a reaction solution that amplifies a nucleic acid having a predetermined sequence and a capture unit having disposed on the surface a first capture substance capable of capturing the nucleic acid in the reaction solution, and a detection unit capable of detecting a change in a physical characteristic of the surface caused by the interaction of the nucleic acid and the first capture substance.

Description

Detection system, cartridge, detection method, nucleic acid probe manufacturing method, and biosensor manufacturing method

The present disclosure relates to a detection system, a cartridge, a detection method, a method for manufacturing a nucleic acid probe, and a method for manufacturing a biosensor using the probe.

When investigating whether a sample contains a detection target (for example, a nucleic acid), a method may be adopted in which the substance is amplified and then detected (Patent Documents 1 and 2).

Furthermore, in recent years, a detection system has been established in which a DNA fragment sample complementary to a DNA fragment immobilized on a solid phase carrier is hybridized. For example, Patent Document 3 describes a method for manufacturing a DNA chip in which DNA fragments and oligonucleotides are arranged on a solid phase surface.

Japanese Patent Publication 2007-105041 Japanese Unexamined Publication 2020-96626 Japanese Patent Application Publication No. 2001-272402

A detection system according to an aspect of the present disclosure includes an amplification unit that performs an amplification reaction of a nucleic acid in a reaction solution that amplifies a nucleic acid having a predetermined sequence, and a first capture substance that can capture the nucleic acid in the reaction solution. a reaction section having a capture section disposed on a surface thereof, and a detection device capable of detecting a change in physical properties of the surface due to interaction between the nucleic acid and the first capture substance.

A detection system according to one aspect of the present disclosure includes a translation part in which a translation reaction from the nucleic acid is performed in a reaction solution containing a nucleic acid having a predetermined sequence and ribosomes, and a translation part capable of capturing translation products in the reaction solution. a reaction unit having a capture unit having a second capture substance disposed on its surface; and a detection device capable of detecting a change in the physical property of the surface due to the interaction between the translation product and the second capture substance. Be prepared.

A cartridge according to an aspect of the present disclosure is a reaction section that accommodates a reaction solution for amplifying a nucleic acid having a predetermined sequence, and includes an amplification section that performs an amplification reaction of the nucleic acid in the reaction solution; a reaction part having a capture part on the surface of which a first capture substance capable of capturing the nucleic acid; and a change in the physical properties of the surface due to the interaction between the nucleic acid and the first capture substance can be detected. and a mounting portion for mounting on a detection device.

A cartridge according to one aspect of the present disclosure includes a reaction section that stores a liquid containing a target nucleic acid having a predetermined sequence, and a capturing section that has a capture substance on a surface that can capture the target nucleic acid in the liquid. and an attachment part for attaching to a detection device capable of detecting a change in the physical properties of the surface due to the interaction between the target nucleic acid and the first capture substance, and the capture substance is , a first capture region including a first nucleic acid fragment having a first complementary sequence complementary to the base sequence of the first end of the target nucleic acid, and a second end different from the first end of the target nucleic acid. and a second capture region including a second nucleic acid fragment having a second complementary sequence complementary to the base sequence of It is a sensor that uses either Surface Plasmon Resonance) or FET (Field Effect Transistor).

A cartridge according to one aspect of the present disclosure is a reaction section that accommodates a reaction solution containing a nucleic acid having a predetermined sequence and a ribosome, and a translation section that performs a translation reaction from the nucleic acid in the reaction solution; a reaction part having a second capture substance on its surface that is capable of capturing a translation product in a reaction solution; and physical properties of the surface resulting from the interaction between the translation product and the second capture substance. and a mounting part for mounting on a detection device capable of detecting a change in the temperature.

A detection method according to an aspect of the present disclosure includes: an amplification unit that performs an amplification reaction of a nucleic acid in a reaction solution that amplifies a nucleic acid having a predetermined sequence; and a first capture substance that can capture the nucleic acid in the reaction solution. an amplification step of carrying out an amplification reaction of the nucleic acid in the reaction solution accommodated in the reaction section; and detecting a change in a physical property of the surface due to the interaction with the first capture substance.

A detection method according to one aspect of the present disclosure includes a translation part in which a translation reaction from the nucleic acid is performed in a reaction solution containing a nucleic acid having a predetermined sequence and a ribosome, and a translation part capable of capturing a translation product in the reaction solution. 2. A containing step of accommodating the reaction solution in a reaction section having a capture section having a capture substance disposed on its surface, and a translation step of carrying out a translation reaction from the nucleic acid in the reaction solution accommodated in the reaction section. and a detection step of detecting a change in physical properties of the surface due to the interaction between the translation product and the second capture substance.

A method for producing a nucleic acid probe according to one aspect of the present disclosure includes a preparation step of preparing a nucleic acid fragment having a base sequence that binds to a target molecule to be captured; and a modification step of modifying at least a portion of the material by adding an electrically neutral functional group or a positively charged functional group.

1 is a diagram showing the appearance of a detection system according to Embodiment 1 of the present disclosure. FIG. 2 is a schematic diagram schematically showing the internal configuration of a cartridge. FIG. 2 is a schematic diagram schematically showing the internal configuration of a sensor unit. It is a figure showing an example of reaction in a reaction part step by step. FIG. 2 is a block diagram showing the main components of a cartridge and a detection device. It is a flowchart which shows an example of the detection method performed in a detection system. It is a figure showing an example of reaction in a reaction part step by step. It is a figure showing an example of reaction in a reaction part step by step. It is a figure showing an example of reaction in a reaction part step by step. It is a flowchart which shows another example of the detection method performed in a detection system. FIG. 2 is a diagram illustrating an example of the configuration of a capture unit including a nucleic acid probe according to one embodiment of the present disclosure. FIG. 2 is a schematic diagram schematically showing an example of an internal configuration of a sensor unit according to one aspect of the present disclosure. FIG. 2 is a schematic diagram schematically showing an example of the configuration of a capturing section according to one aspect of the present disclosure. It is a figure showing an example of the manufacturing method of the trapping part concerning one aspect of the present disclosure. FIG. 2 is a diagram showing an example of addition of a functional group to a phosphate ester included in a nucleic acid fragment according to an embodiment of the present disclosure. FIG. 3 is a diagram illustrating an example of a bond between a fixing part and a polymer according to one embodiment of the present disclosure. FIG. 3 is a diagram illustrating an example of terminal activation of a polymer according to one embodiment of the present disclosure. FIG. 3 is a diagram illustrating a change in charge of a polymer layer due to terminal activation of a polymer according to an embodiment of the present disclosure. FIG. 3 is a diagram showing an example of binding between a nucleic acid probe and a polymer according to one embodiment of the present disclosure. FIG. 3 is a diagram illustrating an example of terminal deactivation of a polymer according to one embodiment of the present disclosure. FIG. 2 is a diagram showing an example of the overall configuration of a capture unit including a nucleic acid probe in which a phosphate ester of a nucleic acid fragment is not modified.

[Embodiment 1]
Hereinafter, one embodiment of the present disclosure will be described in detail.

<Technical thought of this disclosure>
First, the technical concept of the amplification product detection system according to an embodiment of the present disclosure will be described.

For example, an example of a virus detection method that can be used in medical institutions and the like is a PCR test using a PCR method (Polymerase Chain Reaction) to amplify virus-derived nucleic acids.

In order to confirm the amplification product amplified by PCR method or the like or the progress of the amplification reaction, a method is generally designed in which the nucleic acid that is the amplification product is labeled with a fluorescent dye. However, fluorescent dyes are often unstable and must be handled with care, including storage. In place of detection methods that use optical systems, there has been a demand for detection methods that can ensure sufficient detection sensitivity and that can be performed easily.

Therefore, the inventors conducted studies to solve the above problems, and as a result, they developed a detection system, a cartridge, and a detection method that can easily detect amplified products with high sensitivity. The developed detection system, cartridge, and detection method are applicable to the detection of peptides, proteins, etc. in addition to nucleic acids.

<External appearance of detection system 100>
FIG. 1 is a diagram showing the appearance of a detection system 100 according to an embodiment. The detection system 100 includes, as an example, a detection device 3 and a cartridge 2 (flow path device). FIG. 1 shows a state in which the cartridge 2 is not completely attached to the detection device 3 but is being inserted.

In the present embodiment, as an example, in the detection system 100, the detection device 3 and the cartridge 2 are configured as separate bodies, and detection is performed by inserting the cartridge 2 into the detection device 3 and electrically connecting each other. shall be carried out. In such a detection system 100, fluids such as reagents and sample liquid used for detection may be stored in the cartridge 2 in advance, as will be described later. When the detection device 3 and the cartridge 2 are configured as separate bodies, the cartridge 2 is a consumable item, and for example, one cartridge 2 is used to perform detection for one individual subject. In other examples, multiple cartridges 2 may be used per individual subject depending on the type of detection.

Such a configuration of the detection system 100 may be adopted, for example, when performing a rapid test called POCT (Point Of Care Testing), which performs detection immediately after obtaining a sample and presents the result to the user. . In this case, the detection device 3 may be configured as a relatively small device that can be placed in a pharmacy, clinic, home, etc., for example.

The detection system 100 of the present disclosure is not limited to the above-mentioned example, but can also be applied to cases where a large amount of specimens once collected in a laboratory or the like are simultaneously measured using the large detection device 3. In this case, a configuration may be adopted in which fluids such as reagents and sample liquids are sent into the cartridge from the detection device 3 and then into the cartridge when performing detection.

The detection system 100 according to one embodiment is a system that detects a detection target included in a sample P and presents the detection results to a user. The user may be an operator of the detection device 3 in charge of the detection work, a client such as a doctor who requested the measurement, or a subject such as a patient who provided the specimen P. .

The detection system 100 includes, as an example, a cartridge 2 and a detection device 3. After the cartridge 2 is inserted into the detection device 3 and the two are electrically connected, the detection device 3 may start measuring the cartridge 2 immediately, or it may start the measurement according to the user's input operation. good. In another example, the detection device 3 may authenticate the cartridge 2 and start detection if the authentication is successful. The detection device 3 may include a display unit 35 and may present various information to the user. For example, the detection device 3 can display on the display unit 35 the progress of the measurement, a message prompting the user to perform some input operation, an error message when detection is not possible, and the like.

In the detection system 100, the specimen P may be saliva, for example. In the following, the detection system 100 will be described as a system for detecting an amplified substance to be detected contained in saliva as a specimen P. However, the specimen P is not limited to saliva, and may be any substance derived from a living body. The specimen P may be, for example, urine, blood, sweat, or nasal discharge. The cartridge 2 may be configured as appropriate so that the detection device 3 can detect the sample P contained in the cartridge 2. An example of the configuration of the cartridge 2 will be described in detail later with reference to separate drawings.

(Configuration of cartridge 2)
FIG. 2 is a schematic diagram schematically showing the internal configuration of the cartridge 2. As shown in FIG. The cartridge 2 may be a disposable cartridge that can be attached to and detached from the detection device 3. The cartridge 2 may be made of resin, for example. The resin may be, for example, polycarbonate, cycloolefin polymer, polymethyl methacrylate resin, polydimethylsiloxane, and the like.

The cartridge 2 according to one embodiment includes a holding part 21 (a first accommodating part, a second accommodating part), a liquid receiving part 22 (a first accommodating part, a second accommodating part), a sensor unit 23, and a flow path 28. and an attachment part 29 for attaching the cartridge to the detection device 3.

The holding unit 21 holds a liquid, particularly a liquid such as a reagent that does not contain a measurement target. In this embodiment, as an example, the detection system 100 may use two different types of reagents for measurement. Hereinafter, the holding section 21 that holds the first reagent will be referred to as a first holding section 211 (first storage section, second storage section), and the holding section 21 that holds the second reagent will be referred to as a second holding section 212 ( 1st storage part, 2nd storage part). In other examples, the detection system 100 may use one type of reagent for measurement, or may use three or more types of reagents for measurement. That is, the cartridge 2 may include one holding section 21, or may include three or more holding sections 21. For example, when amplifying nucleic acids, the holding section 21 accommodates enzymes necessary for amplifying the nucleic acids. Examples of enzymes necessary for nucleic acid amplification include DNA polymerase and RNA polymerase.

The liquid receiving part 22 takes in a liquid, particularly a sample P as a sample liquid containing a measurement target, into the cartridge 2 and holds it therein. The shape of the liquid receiving part 22 is not particularly limited. The liquid receiving portion 22 is connected to a flow path 28 . The sample P accommodated in the liquid receiving part 22 is pushed out from the liquid receiving part 22 by being pressed by a pressing pin (not shown) of the detection device 3, and is supplied to the sensor unit 23 via the connected flow path 28. be done. The liquid receiving part 22 may be formed integrally with the flow path 28 or may be formed separately from the flow path 28. The liquid receiving portion 22 may be formed by a conventionally known technique.

The sensor unit 23 detects a measurement target included in the sample P. The sensor unit 23 includes at least one sensor that detects a measurement target. The sensor unit 23 may include multiple sensors. As an example, the sensor unit 23 may include two sensors: a reaction section 50 (sensor, first sensor) and a reference section 25 (sensor, second sensor). Below, when the sensor unit 23 has a single sensor, the sensor unit 23 may be simply referred to as a sensor. When the sensor unit 23 has a plurality of sensors, for example, the reaction section 50 and the reference section 25, the reaction section 50 and the reference section 25 may be collectively referred to as a sensor if there is no need to distinguish between the individual sensors. good. Further, the entire sensor unit 23 including the reaction section 50 and the reference section 25 may be referred to as a sensor.

The reaction section 50 includes an amplification section 51 that performs a nucleic acid amplification reaction in a reaction solution that amplifies a nucleic acid having a predetermined sequence, and a capture section 24 that has a first capture substance on its surface that can capture the nucleic acid in the reaction solution. and has. In this disclosure, "surface" is intended to be the surface on which the first capture substance is placed. The reaction section 50 is a place where amplification reaction and capture are performed, and if the amplification reaction and capture are separated or limited, the reaction section 50 is a place where the amplification reaction and capture are performed, even if they are at a specific location. good. When the place where the amplification reaction proceeds is not limited, the reaction section 50 can be any place where the reaction solution is accommodated. For example, when the amplification reaction and the capture are performed at a predetermined location in the channel 28, the predetermined location may be the reaction section 50.

The capture unit 24 captures nucleic acids in the reaction solution. For example, if a primer for amplifying a nucleic acid is present on the surface of the capture section 24, an amplification reaction may proceed in the capture section 24. The capture unit 24 will be described later.

The flow path 28 is for supplying one or more types of fluid to the sensor unit 23. The flow path 28 extends inside the cartridge 2 so as to connect each of the above-mentioned components of the cartridge 2, specifically, the first holding part 211, the second holding part 212, and the liquid receiving part 22, and the sensor unit 23. is formed. The fluid contained in the first holding part 211, the second holding part 212, and the liquid receiving part 22 is supplied to the sensor unit 23 via the flow path 28.

The attachment part 29 is a mechanism for attaching and fixing the cartridge 2 to the detection device 3. Further, the attachment portion 29 electrically connects the cartridge 2 and the detection device 3. Since the cartridge 2 includes the attachment portion 29, the cartridge 2 is attached to the detection device 3, and the cartridge 2 and the detection device 3 are electrically connected.

As described above, the cartridge 2 can be electrically connected to the detection device 3 and can input and output electrical signals to and from the detection device 3. Terminals and the like for electrically connecting the cartridge 2 and the detection device 3 may be manufactured by a conventionally known method. In other examples, the cartridge 2 may not be physically attached to the detection device 3. For example, the cartridge 2 may include a communication section capable of communicating with the detection device 3. In this case, the cartridge 2 may transmit and receive various information such as electrical signals related to the test to and from the detection device 3 through wired or wireless communication.

(Configuration of sensor unit 23)
FIG. 3 is a schematic diagram schematically showing the internal configuration of the sensor unit 23. As shown in FIG. The sensor unit 23 according to one embodiment is, for example, a sensor using elastic waves, and includes a reaction section 50, a reference section 25, a pair of first IDT (Inter Digital Transducer) electrodes 26A, a pair of second IDT electrodes 26B, and A substrate 27 is provided. The reaction section 50, the reference section 25, the pair of first IDT electrodes 26A, and the pair of second IDT electrodes 26B may be located on the substrate 27.

The reaction section 50 and the reference section 25 of the sensor unit 23 may be sensors that utilize elastic waves, QCM (Quartz Crystal Microbalance), SPR (Surface Plasmon Resonance), FET (Field Effect Transistor), or the like, for example. That is, the sensor unit 23 may mutually convert an electric signal and an elastic wave, QCM, SPR, FET, etc. The sensor unit 23 may be manufactured by a conventionally known method. As described above, the sensor unit 23 according to one embodiment is, for example, a sensor device that uses elastic waves, and can mutually convert an electric signal into an elastic wave and an elastic wave into an electric signal. In this case, information specific to a sensor using elastic waves, such as the initial phase of the elastic waves and the orientation of the substrate 27, may be held in the detection device 3.

The capture unit 24 has a capture substance capable of capturing the amplification product in the reaction solution on its surface. The capture substance will be described later. The surface of the trap 24 may be made of metal, for example. Specifically, the trap 24 may be made of metals such as gold, chromium, and titanium, or a combination of these metals. The trapping section 24 may be a single-layer metal film made of a single material, or a multilayer metal film made of a plurality of materials. The material constituting the trapping section 24 is not limited to the above-mentioned metals, but any material that has a function of fixing the trapped substance can be used. The trap 24 may be manufactured by a conventionally known method.

The capture substance can be, for example, a fragment of a nucleic acid, an antibody, an enzyme, etc. That is, the detection target may be, for example, a nucleic acid, an antigen, a substrate, a protein, or the like. Detection targets are not limited to these examples. In this way, depending on the purpose, the detection target to be detected by the detection device 3 may be selected as appropriate, and the capture substance paired with the detection target may be selected as appropriate. In this embodiment, a case will be described in which the capture substance is a fragment of a nucleic acid and the detection target is a nucleic acid.

The pair of first IDT electrodes 26A can generate elastic waves between the pair of first IDT electrodes 26A. Among the generated elastic waves, the elastic waves that propagate on the surface of the substrate 27 are also referred to as surface acoustic waves (SAW). The pair of first IDT electrodes 26A may be arranged on the substrate 27 so as to sandwich the reaction section 50 therebetween. In the sensor unit 23 according to one embodiment, an electric signal (input signal) is input to one of the pair of first IDT electrodes 26A under the control of the detection device 3. The input electrical signal is converted into an elastic wave that propagates toward the reaction section 50 and is transmitted from one first IDT electrode 26A. The emitted elastic waves pass through the reaction section 50. The other first IDT electrode 26A can receive the elastic wave that has passed through the reaction section 50. The received elastic waves are converted into electrical signals (output signals). The converted electrical signal is output to the detection device 3. The pair of first IDT electrodes 26A may be formed of a metal such as gold, chromium, or titanium, or a combination of these metals. The pair of first IDT electrodes 26A may be a single layer electrode made of a single material, or a multilayer electrode made of a plurality of materials.

In the capture unit 24 included in the reaction unit 50, the interaction between the nucleic acid and the capture substance changes the propagation characteristics of the elastic wave propagating on the substrate 27. Specifically, the reaction between the nucleic acid and the capture substance changes the weight applied to the substrate 27 or the viscosity of the liquid that contacts the surface of the substrate 27. The magnitude of these changes correlates with the amount of reaction between the nucleic acid and the capture substance. Furthermore, the characteristics of the elastic wave (for example, phase, amplitude, period, etc.) change as it propagates through the capture unit 24. The magnitude of the change in characteristics correlates with the magnitude of the weight applied to the substrate 27 or the magnitude of the viscosity of the liquid that contacts the surface of the substrate 27. Therefore, the detection device 3 can detect nucleic acids based on changes in the propagation characteristics of elastic waves by analyzing the output signal output from the sensor unit 23. Specifically, the detection device 3 can calculate, for example, the concentration of the amplified nucleic acid.

The sensor unit 23 may have two or more combinations of the capturing section 24 and the pair of IDT electrodes 26A. In this case, the detection device 3 may measure different types of target substances for each combination, for example. Alternatively, the detection device 3 may, for example, measure the same type of target substance in multiple combinations and compare the respective measurement results.

Unlike the trapping portion 24, the reference portion 25 does not have a captured substance fixed thereto. Therefore, in the reference section 25, no reaction occurs between the nucleic acid and the capture substance. Therefore, the reference section 25 can function as a control for the capture section 24. The reference section 25 may be configured the same as or similar to the capture section 24 .

The pair of second IDT electrodes 26B can generate elastic waves between the pair of second IDT electrodes 26B. The pair of second IDT electrodes 26B may be arranged on the substrate 27 so as to sandwich the reference portion 25 therebetween. In the sensor unit 23 according to one embodiment, an electrical signal (input signal) is input to one of the pair of second IDT electrodes 26B under the control of the detection device 3. The input electrical signal is converted into an elastic wave that propagates toward the reference section 25 and is emitted from one second IDT electrode 26B. The emitted elastic wave passes through the reference section 25. The other second IDT electrode 26B can receive the elastic wave that has passed through the reference section 25. The received elastic waves are converted into electrical signals (output signals). The converted electrical signal is output to the detection device 3. The pair of second IDT electrodes 26B may be configured the same as or similar to the pair of first IDT electrodes 26A.

The substrate 27 may be a piezoelectric substrate, for example. As an example, the substrate 27 may be a crystal substrate. The substrate 27 is not limited to a crystal substrate, and may be made of any material capable of propagating elastic waves. The substrate 27 may be manufactured by a conventionally known method.

(Reaction in reaction section 50)
FIG. 4 is a cross-sectional view taken along the line III-III of the reaction section 50 in FIG. 3, and is a diagram showing the reaction in the reaction section 50 (amplification reaction in the amplification section 51 and capture in the capture section 24) step by step. The reaction section 50 includes an amplification section 51 that performs a nucleic acid amplification reaction in a reaction solution that amplifies a nucleic acid having a predetermined sequence contained in the sample P, and a first capture substance on the surface that can capture the nucleic acid in the reaction solution. It has a capturing part 24 arranged therein. In FIG. 4, the amplification reaction is performed in the entire reaction solution in the reaction section 50. Further, in the reaction section 50, a trapping section 24 is arranged on the substrate 27, and a first trapping substance is arranged on the surface of the trapping section 24.

In the reaction section 50 of this embodiment, the nucleic acid amplified in the amplification section 51 is captured by the first capture substance of the capture section 24.

For the amplification reaction in the amplification section 51, a known nucleic acid amplification method may be used. The amplification reaction includes, for example, a heat denaturation step, an annealing step, and a DNA extension step. The amplification reaction may include a plurality of cycles with the above steps as one cycle. The amplification reaction progresses as appropriate under temperature control by the temperature control section 46.

The reaction section 50 may include the capture section 24 in the same system as the amplification section 51. Furthermore, in the reaction section 50, the amplification reaction and the capture of the nucleic acid in the reaction solution by the first capture substance may be performed simultaneously. "Performed simultaneously" means that the amplification reaction and the capture (reaction) of the nucleic acid should be performed in parallel for most of the time period from the start to the end of each reaction. Strictly speaking, the start of the amplification reaction and the time when the nucleic acid is first captured are not simultaneous, but the start of either the amplification reaction or the capture is used as a trigger to start the other reaction. That's fine.

The detection system 100 includes an amplification section 51 in which nucleic acids are amplified, and a capture section 24 that has a first capture substance on its surface that can capture nucleic acids in the reaction solution. Amplification and nucleic acid capture can be performed within the same system.

Furthermore, in the reaction section 50, the amplification reaction and the capture of the nucleic acid in the reaction solution by the first capture substance are performed simultaneously, so that the time from amplification to detection can be shortened. That is, the time from when the user attaches the sample P to the cartridge to when the detection result is obtained is shortened.

The first capture substance may be capable of capturing at least one of a nucleic acid that serves as a template in an amplification reaction and a nucleic acid that is an amplification product from the amplification reaction. The nucleic acid of an amplification product is a nucleic acid amplified by an amplification reaction. In this embodiment, a case will be described in which the first capture substance is capable of capturing the nucleic acid of the amplification product resulting from the amplification reaction.

The first capture substance may include a first nucleic acid fragment having a first complementary sequence that is complementary to the base sequence at the first end of the amplification product. Specifically, the first end portion of the amplification product is DNA, RNA, LNA, BNA, or μRNA. Furthermore, the first capture substance may be modified at its 3' end, for example, so that it does not extend itself.

In FIG. 4, a mode is shown in which a pair of fragments, a first nucleic acid fragment F1 having a sequence A' and a first nucleic acid fragment F11 having a sequence D, are arranged as a first capture substance on the surface of the capture part 24. show. FIG. 4 only schematically shows the trapping section 24, and a plurality of first trapping substances may be arranged on the surface of the trapping section 24. The number of first capture substances disposed in the capture unit 24 is not particularly limited, but for example, the number of first capture substances arranged in the capture unit 24 is such that the amplification product can be captured to the extent that a change in the physical properties of the surface of the capture unit 24 can be detected by the detection device 3. A trapping substance may be provided.

For example, in step (1) in FIG. 4, the nucleic acid is first amplified in the amplification section 51. In step (2), the amplified nucleic acid becomes single-stranded by heat denaturation. Then, the single-stranded nucleic acid is captured by a first nucleic acid fragment F11 having a first complementary sequence (sequence D') that is complementary to the base sequence (sequence D) at the first end of the nucleic acid. . In step (3), the other nucleic acid that has become single stranded in step (2) has a first complementary sequence (sequence A) that is complementary to the base sequence (sequence A) at the first end of the nucleic acid. ') is captured by the first nucleic acid fragment F1. During steps (1) to (3), the amplification reaction in the amplification section 51 is progressing, and each time the amplification product is annealed, the nucleic acid that is the amplification product and the first nucleic acid fragment that is the first capture substance are complementary to each other. join to.

<Configuration of detection system 100>
FIG. 5 is a block diagram showing the main components of the cartridge 2 and the detection device 3 that constitute the detection system 100. As described above, the detection system 100 includes the cartridge 2 and the detection device 3.

As described above, the press pins (not shown) of the connected detection device 3 are pushed down toward the liquid receiving part 22, the first holding part 211, and the second holding part 212 of the cartridge 2, respectively. Accordingly, the liquid stored inside each of the liquid receiving part 22, the first holding part 211, and the second holding part 212 is pushed out to the flow path 28, and passes through the flow path 28 to the sensor unit 23. supplied to

(Hardware configuration of detection device 3)
The detection device 3 is a device capable of detecting changes in the physical characteristics of the trapping section 24 due to interaction with the first trapping substance described above. The physical property detectable by the detection device may be at least one of surface acoustic waves, frequency, refractive index, and conductivity.

The detection device 3 includes, for example, a control section 31, a storage section 32, a pressing section 33, a signal processing section 34, a display section 35, a communication section 36, and a temperature adjustment section 37.

The control section 31 centrally controls each section of the detection device 3. The control unit 31 is configured by, for example, an arithmetic device such as a CPU (Central Processing Unit) or a dedicated processor. Each part of the control unit 31, which will be described later, allows the above-mentioned arithmetic unit to read a program stored in a storage device (for example, the storage unit 32) implemented using a ROM (read only memory) or the like into a RAM (random access memory) or the like. This can be achieved by executing

Additionally, the control section 31 may include a temperature control section 46 for controlling the temperature of the temperature adjustment section 37. The temperature control section 46 controls the temperature adjustment section 37 to raise and lower the temperature of the reaction section 50 according to a preset program.

The storage unit 32 stores various data processed by the control unit 31 and various data referred to during processing. As an example, the storage unit 32 stores a normal model. The normal model is referred to by the control unit 31 when the control unit 31 determines whether there is a measurement defect.

The pressing part 33 is a drive mechanism for pushing out liquid from each of the liquid receiving part 22, the first holding part 211, and the second holding part 212. The pressing section 33 includes, for example, a pressing pin (not shown) and an actuator that generates power for pressing the pressing pin down toward the cartridge 2 .

The signal processing section 34 transmits and receives electrical signals to and from the electrically connected sensor unit 23. When the electrical signal to be analyzed for measurement is the output signal OS output from the sensor unit 23, the signal processing section 34 receives at least the output signal OS from the sensor unit 23. When analysis is performed using both the input signal IS input to the sensor unit 23 and the above-mentioned output signal OS, the signal processing section 34 further generates the input signal IS under the control of the control section 31. , is transmitted to the sensor unit 23.

The display unit 35 outputs various data processed by the control unit 31 as visual information that can be viewed by the user.

The communication unit 36 is configured by wireless or wired communication means, and communicates with other devices. In the detection system 100 in which the detection device 3 does not need to communicate with an external device, the communication unit 36 may be omitted.

The detection device 3 may further include an operation unit that accepts input operations from the user. The operating section may be configured as hardware components such as buttons and switches, or may be configured as a combination of a touch panel formed integrally with the display section 35 and software components displayed on the display section 35.

The temperature adjustment section 37 can adjust the temperature of the reaction section 50 of the cartridge 2, which will be described later, based on the control of the temperature control section 46. The temperature adjustment section 37 may be a heater or a cooler, although it is not particularly limited, and the temperature adjustment section 37 may be configured by, for example, a compressor, a Peltier element (heat transfer module), a heat lid, or the like.

<Detection method>
FIG. 6 is a flowchart showing a process flow of a detection method executed by the detection system 100 according to an embodiment of the present disclosure.

In step S1, the control unit 31 of the detection device 3 first detects that the cartridge 2 is electrically connected to the detection device 3. In detection system 100 in which cartridge 2 is not provided, this step may be omitted. When detecting that the cartridge 2 is connected, the control unit 31 advances the process from YES in S1 to S2.

In step S2, an amplification unit 51 that performs a nucleic acid amplification reaction in a reaction solution that amplifies a nucleic acid having a predetermined sequence, and a capture unit 24 that has a first capture substance on its surface that can capture nucleic acids in the reaction solution. A reaction solution is accommodated in the reaction section 50 having the following (accommodation step).

In step S3, a nucleic acid amplification reaction is performed in the reaction solution contained in the reaction section 50 (amplification step). Specifically, the temperature controller 46 adjusts the temperature of the temperature controller 37 according to a temperature adjustment program for amplifying nucleic acids.

In step S4, the detection device 3 detects a change in the physical properties of the surface due to the interaction between the nucleic acid and the first capture substance (detection step). The detection step may be started before the amplification reaction in the amplification step is completed, and the amplification step and the detection step may be performed simultaneously. That is, the trigger for starting the detection step may be the presence of a nucleic acid amplified by the amplification reaction of the amplification step, but in an amplification reaction that can be performed in multiple cycles, detection is performed at the end of one cycle. Steps may be initiated.

The detection method of the present embodiment includes an accommodation step of accommodating a reaction solution in a reaction section 50 having an amplification section 51 and a capture section 24, an amplification step of performing an amplification reaction in the reaction section 50, a nucleic acid, and a first capture section. and detecting changes in physical properties of the surface due to interaction with the substance. According to the above method, the amplification reaction and the capture can be performed simultaneously in the same system, so the time from the start of the amplification reaction to detection is shortened. Further, according to the above-described method, since changes in the physical properties (for example, propagation properties of elastic waves) of the surface of the capture unit 24 are utilized for detection, the detection system 100 detects the amplified nucleic acid in the reaction solution with high sensitivity. It can be detected easily and easily.

[Embodiment 2]
Other embodiments of the present disclosure will be described below. For convenience of explanation, members having the same functions as those described in the above embodiments are denoted by the same reference numerals, and the description thereof will not be repeated.

In Embodiment 1, the nucleic acid amplified in the amplification unit 51 was captured in the capture unit 24, but in this embodiment, the amplification reaction proceeds in the capture unit 24A, which is different from Embodiment 1. different from. That is, in this embodiment, the amplifying section 51A and the capturing section 24A are the same.

FIG. 7 is a diagram showing the reaction in the reaction section 50A (the amplification reaction in the amplification section 51A and the capture in the capture section 24A) step by step.

The amplifying section 51A may have a surface on which the first capturing substance is disposed and functioning as the capturing section 24A. This means that the amplifying section 51A also has a function as the capturing section 24A. According to this, in the detection system 100, the amplification reaction progresses in the capture unit 24A, and the detection device 3 can detect changes over time in the amplification reaction.

In this embodiment, the first capture substance may include a second nucleic acid fragment having a second complementary sequence that is complementary to the base sequence at the second end of the nucleic acid serving as a template. Furthermore, the length of the second nucleic acid fragment that has captured the template nucleic acid is extended by an amplification reaction using the captured template nucleic acid as a template.

In FIG. 7, a pair of fragments, a second nucleic acid fragment F2 having the sequence A' and a second nucleic acid fragment F21 having the sequence D, are arranged as the first trapping substance on the surface of the trapping part 24A. shows. The second nucleic acid fragment F2 and the second nucleic acid fragment F21 may be primers.

For example, in step (1) of FIG. 7, the nucleic acid T serving as a template is separated from a double-stranded state into a single-stranded one by thermal denaturation. In step (2), the single-stranded nucleic acid T1 and nucleic acid T2 are captured by the second nucleic acid fragment F2 and the second nucleic acid fragment F21, respectively. Specifically, the nucleic acid T1 binds to a second nucleic acid fragment F2 having a second complementary sequence (sequence A') that is complementary to the base sequence (sequence A) at the second end of the nucleic acid T1. Furthermore, the nucleic acid T2 binds to a second nucleic acid fragment F21 having a second complementary sequence (sequence D) that is complementary to the base sequence (sequence D') at the second end of the nucleic acid T2.

In step (3), the second nucleic acid fragments F2 and F21 to which nucleic acid T1 and nucleic acid T2 are complementary bound are elongated using nucleic acid T1 and nucleic acid T2 as templates, respectively, and new nucleic acid T1' and nucleic acid T2' are amplified. (amplification reaction).

In step (4), nucleic acid T1' and nucleic acid T2' become single-stranded by heat denaturation. As a result, the amplified nucleic acid is immobilized on the capture portion 24A.

In this way, since the amplification section 51A has the function of the capture section 24A, the detection system 100 not only detects the nucleic acid of the amplification product, but also detects changes over time in the amplification reaction on the surface of the capture section 24A. It can be detected as a change in characteristics. That is, the detection system 100 can detect the progress of the amplification reaction in real time.

[Embodiment 3]
Other embodiments of the present disclosure will be described below. For convenience of explanation, members having the same functions as those described in the above embodiments are denoted by the same reference numerals, and the description thereof will not be repeated.

This embodiment is similar to Embodiment 2 in that the amplification reaction proceeds in the capture section 24B, but is similar to Embodiment 2 in that the end of the nucleic acid chain extended by the amplification reaction is further captured in the capture section 24B. It is different from.

FIG. 8 is a diagram showing the reaction in the reaction section 50B (the amplification reaction in the amplification section 51B and the capture in the capture section 24B) step by step.

Similarly to Embodiment 2, the amplification section 51B may include, as the first capture substance, a second nucleic acid fragment having a second complementary sequence that is complementary to the base sequence at the second end of the nucleic acid serving as a template. In this embodiment, the first capture substance is a third compound having a third complementary sequence that is complementary to the base sequence of the third end located far from the surface of the nucleic acid strand extended by the amplification reaction. It may further contain a nucleic acid fragment.

In FIG. 8, a pair of fragments, a second nucleic acid fragment F2 having the sequence A' and a second nucleic acid fragment F21 having the sequence D, are arranged as the first trapping substance on the surface of the trapping part 24B. Furthermore, in addition to the second nucleic acid fragments F2 and F21, a third nucleic acid fragment F3 having sequence D and a third nucleic acid fragment F31 having sequence A' are arranged on the surface side of the capture portion 24B. FIG. 8 shows, as an example, a mode in which the second nucleic acid fragment F2 (F21) and the third nucleic acid fragment F3 (F31) are connected as a single chain. However, the second nucleic acid fragment F2 (F21) and the third nucleic acid fragment F3 (F31) do not necessarily have to be a single strand; for example, the second nucleic acid fragment F2 (F21) and the third nucleic acid fragment F3 (F31) may be arranged separately on the surface side of the capturing portion 24B. In this case, as will be described later, the second nucleic acid fragment F2 (F21) and the third nucleic acid fragment F3 (F31) may be arranged at a close distance to such an extent that the amplified nucleic acid can form a folded structure. The second nucleic acid fragment F2 and the second nucleic acid fragment F21 may be primers.

For example, in step (1) of FIG. 8, the nucleic acid T serving as a template is separated from a double-stranded state into a single-stranded one by thermal denaturation. In step (2), the single-stranded nucleic acid T1 and nucleic acid T2 are captured by the second nucleic acid fragment F2 and the second nucleic acid fragment F21, respectively. Specifically, the nucleic acid T1 binds to a second nucleic acid fragment F2 having a second complementary sequence (sequence A') that is complementary to the base sequence (sequence A) at the second end of the nucleic acid T1. Furthermore, the nucleic acid T2 binds to a second nucleic acid fragment F21 having a second complementary sequence (sequence D) that is complementary to the base sequence (sequence D') at the second end of the nucleic acid T2.

In step (4), nucleic acid T1' and nucleic acid T2' become single-stranded by heat denaturation. As a result, the amplified nucleic acid is immobilized on the capture portion 24A. Further, the third end portion of the nucleic acid T1′ on the side far from the capture portion 24B is a third nucleic acid fragment having a third complementary sequence (sequence D) that is complementary to the base sequence (sequence D′) of the third end portion. Captured by F3. The third end of the nucleic acid T2' far from the capture portion 24B is a third nucleic acid fragment F31 having a third complementary sequence (sequence A') that is complementary to the base sequence (sequence A) of the third end. Captured. That is, one nucleic acid has two ends fixed to the capture portion 24B in a folded structure.

In this way, since the amplification section 51B has the function of the capture section 24B, the detection system 100 not only detects the nucleic acid of the amplification product, but also detects changes over time in the amplification reaction on the surface of the capture section 24B. It can be detected as a change in characteristics. That is, the detection system 100 can detect the progress of the amplification reaction in real time.

Furthermore, since the capture portion 24B includes the third nucleic acid fragment, the surface characteristics of the capture portion 24B change more greatly when nucleic acid is captured than in the capture portion 24A of the second embodiment. In particular, in the case of a sensor in which the capture section 24B uses SAW, when two ends of the nucleic acid are captured by the capture section 24B, the capture section The surface density of 24B increases, resulting in a higher surface viscosity. Even when the amount of amplified nucleic acid is small, the detection system 100 can detect the nucleic acid of the amplification product with high sensitivity.

[Modified example]
Modifications of the present disclosure will be described below. For convenience of explanation, members having the same functions as those described in the above embodiments are denoted by the same reference numerals, and the description thereof will not be repeated.

In Embodiments 1 to 4, the

reaction sections

50, 50A, and 50B each included the

capture sections

24, 24A, and 24B, and the

amplification sections

51, 51A, and 51B. On the other hand, in this modification, the reaction section 50 includes only the capture section 24. That is, in Embodiments 1 to 3, the capture section 24 and the amplification section 51 were provided in the same system, and the amplification reaction of the nucleic acid and the capture of the nucleic acid were performed simultaneously, but in this modification, Nucleic acid amplification reactions and nucleic acid capture are performed at different locations.

In this modification, the cartridge 2 includes a reaction section 50 and an attachment section 29. The reaction section 50 contains a liquid containing a target nucleic acid having a predetermined sequence, and has a capturing section 24 on the surface of which a capture substance capable of capturing the target nucleic acid in the liquid is disposed. Furthermore, the attachment section 29 is a mechanism for attaching the target nucleic acid to the detection device 3 capable of detecting changes in the physical properties of the surface caused by the interaction with the capture substance.

The capture substance includes a first capture region that includes a first nucleic acid fragment having a first complementary sequence complementary to the base sequence of the first end of the target nucleic acid, and a second end that is different from the first end of the target nucleic acid. and a second capture region containing a second nucleic acid fragment having a second complementary sequence that is complementary to the base sequence of the second region.

According to this, the detection system 100 uses the capture unit 24 to capture the nucleic acid amplified at a location other than the capture unit 24. That is, by dropping a liquid containing the amplified nucleic acid onto the cartridge 2, the detection system 100 can detect the amplified nucleic acid. According to this, the amplified nucleic acid can be detected with high sensitivity regardless of the type of amplification reaction method.

[Embodiment 4]
Other embodiments of the present disclosure will be described below. For convenience of explanation, members having the same functions as those described in the above embodiments are denoted by the same reference numerals, and the description thereof will not be repeated.

Detection of mRNA and μRNA in specimens is required for early testing of various diseases, but detection requires the use of LC-MSMS, next-generation sequencers, etc. Furthermore, since short RNA contained in a sample has a small molecular weight, it may be difficult to detect it even when, for example, a SAW is used as a capture unit. This embodiment performs amplification of RNA that may be contained in such a sample, translation into protein, and detection of protein in the same system.

In Embodiments 1 to 3, the reaction section 50 includes an amplification section 51 and a capture section 24, and the capture section 24 captures nucleic acids. On the other hand, in this embodiment, the reaction section 50C includes a translation section 52 and a capture section 24C, and the capture section 24C captures the translation product generated by the translation reaction.

In this embodiment, the detection device 3 is capable of detecting changes in the physical properties of the surface caused by the interaction between the translation product and a second capture substance, which will be described later.

The reaction section 50C includes a translation section 52 in which a translation reaction from a nucleic acid is carried out in a reaction solution containing a nucleic acid having a predetermined sequence and ribosomes, and a second capture substance on the surface that can capture translation products in the reaction solution. It has a capturing section 24C.

The reaction solution may contain transfer RNA and amino acid molecules. According to this, the protein translation reaction progresses in the translation section 52. That is, in this embodiment, the holding portion 21 of the cartridge 2 accommodates ribosomes, transfer RNA, amino acid molecules, and the like.

The translation unit 52 may perform a protein translation reaction and an amplification reaction in which a nucleic acid used in the translation reaction is amplified before the translation reaction. In the translation section 52, the amplification reaction and the translation reaction may be performed simultaneously. "To be performed simultaneously" means that the amplification reaction and the translation reaction are performed in parallel for most of the time period from the start to the end of the nuclear reaction. Strictly speaking, the start of the amplification reaction and the start of the translation reaction are not simultaneous, but the translation reaction is triggered by the start of the amplification reaction and the amplification of a nucleic acid having a predetermined sequence. Bye. Hereinafter, "translation reaction" will be described as including amplification reaction.

In the reaction section 50C, the translation reaction and the capture of the translation product in the reaction solution by the second capture substance may be performed simultaneously. "Performed simultaneously" means that the translation reaction and the capture (reaction) of the translation product may be performed in parallel for most of the time period from the start to the end of each reaction. Strictly speaking, the start of the translation reaction and the time when the translation product is first captured are not simultaneous, but the capture may be started when a translation product is generated by the translation reaction.

FIG. 9 is a diagram showing the reaction in the reaction section 50C (translation reaction in the translation section 52 and capture in the capture section 24C) step by step.

For example, in step (1) in FIG. 9, a nucleic acid amplification reaction and a translation reaction from the amplified nucleic acid are performed in the translation unit 52, and protein P is generated.

In step (2), protein P produced by the translation reaction is captured by ligand L.

In this way, since the reaction section 50C includes the translation section 52 and the capture section 24C, the detection system 100 not only detects proteins that are translation products, but also detects changes over time produced by translation products. This can be detected as a change in the characteristics of the surface of the trapping section 24C.

In addition, since the target nucleic acid is translated into a protein with a large molecular weight before being detected, the characteristics of the surface of the capture part 24C change more significantly than when directly detecting the nucleic acid, and the translation product can be detected with high sensitivity. can do. Furthermore, the detection system 100 has the effect of making it easier for the user to identify the causative agent by detecting the source (translation product) of diseases such as cancer metastasis and infectious diseases, rather than directly detecting nucleic acids. play.

Furthermore, with the above configuration, the detection system 100 can perform multi-step reactions such as amplification reaction, translation reaction, and capture in the same system and perform them simultaneously. The time it takes to detect is shortened.

<Detection method>
FIG. 10 is a flowchart showing the process flow of a detection method executed by the detection system 100 according to an embodiment of the present disclosure.

In step S11, the control unit 31 of the detection device 3 first detects that the cartridge 2 is electrically connected to the detection device 3. In detection system 100 in which cartridge 2 is not provided, this step may be omitted. When detecting that the cartridge 2 is connected, the control unit 31 advances the process from YES in S1 to S12.

In step S12, a translation unit 52 in which a translation reaction from a nucleic acid is performed in a reaction solution containing a nucleic acid having a predetermined sequence and ribosomes, and a second capture substance capable of capturing translation products in the reaction solution are arranged on the surface. The reaction liquid is accommodated in the reaction section 50C having the captured section 24C (accommodation step).

In step S13, a translation reaction from the nucleic acid is performed in the reaction solution contained in the reaction section 50C (translation step).

In step S14, the detection device 3 detects a change in the physical properties of the surface caused by the interaction between the translation product and the second capture substance (detection step). The detection step may be started before the translation reaction in the translation step is completed, and the translation step and the detection step may be performed simultaneously. That is, the trigger for starting the detection step may be the presence of a translation product translated by the translation reaction of the translation step, but in a translation reaction that may occur sequentially, the detection step starts at the point when one reaction is completed. may be started.

The detection method of this embodiment includes an accommodation step of accommodating a reaction solution in a reaction section 50C having a translation section 52 and a capture section 24C, a translation step of performing a translation reaction, and an interaction between a translation product and a second capture substance. detecting changes in physical properties of the surface due to the action. According to the above method, the translation reaction and the capture can be performed simultaneously in the same system, so the time from the start of the translation reaction to detection is shortened. Further, according to the above-described method, since changes in the physical properties (for example, propagation properties of elastic waves) of the surface of the capture unit 24 are utilized for detection, the detection system 100 detects the amplified nucleic acid in the reaction solution with high sensitivity. It can be detected easily and easily.

[Embodiment 5]
Hereinafter, one embodiment of the capture unit 24 including the nucleic acid probe 243 will be described in detail.

<Overview of Nucleic Acid Probe 243>
First, the outline of the nucleic acid probe 243 according to this embodiment will be explained using FIG. 11. FIG. 11 is a diagram showing an example of the overall configuration of the capture section 24 including the nucleic acid probe 243. The capture unit 24 has a function of specifically capturing target molecules, and can be applied to biosensors, detection systems, etc. that detect target organisms.

As shown in FIG. 11, the capturing section 24 includes a fixing section 241, a polymer layer 242a made of a polymer 242, and a nucleic acid probe 243. The fixing portion 241 and the polymer 242 are bonded at a bonding portion B1. Further, the polymer 242 and the nucleic acid probe 243 are bonded at a bonding portion B2. Details of the fixing portion 241, the polymer 242, the bonding portion B1, and the bonding portion B2 will be described later.

The nucleic acid probe 243 has a base sequence that binds to the target molecule to be captured. Here, the target molecule may be, for example, a nucleic acid, a peptide capable of binding to a nucleic acid, a transcription regulatory factor, a nucleic acid binding protein such as a polymerase, and the like.

Furthermore, as shown in FIG. 11, the base sequence is composed of polynucleotides. The polynucleotide is obtained by polymerizing nucleotides through a phosphodiester bond between the phosphate groups of each nucleotide. Hereinafter, a phosphodiester possessed by a polynucleotide will be referred to as a "phosphate ester." That is, the phosphate ester is a component constituting the nucleic acid probe 243.

Further, at least some of the plurality of phosphoric acid esters constituting the nucleic acid probe 243 have an electrically neutral functional group R or a positively charged functional group R added thereto.

Furthermore, a cation may be coordinated to at least some of the plurality of phosphate esters that constitute the nucleic acid probe 243.

For example, the nucleic acid probe 243 may be electrically neutral or positively charged in an aqueous solution ranging from pH 6 to pH 8.

Here, the advantageous properties of the nucleic acid probe 243 will be explained using FIG. 21. FIG. 21 is a diagram showing an example of the overall configuration of the capture section 1024 including the nucleic acid probe 1243 that is not modified with a phosphate ester. The capturing section 1024 includes a fixing section 1241, a polymer layer 1242a made of a polymer 1242, and a nucleic acid probe 1243. The phosphoric acid ester of nucleic acid probe 1243 has not been subjected to addition of a functional group.

The nucleic acid probe 1243 whose phosphoric acid ester has not been modified such as addition of a functional group will have a negative charge in an aqueous solution. When the nucleic acid probe 1243 is immobilized on the immobilization part 1241 on which the polymer 1242 having a positive electrical property is disposed, the following phenomenon may occur. An electrical attraction is generated between the polymer 1242, which has a positive electrical property, and the nucleic acid probe 1243, which has a negative electrical property, and which is arranged on the fixing part 1241. Therefore, as shown in FIG. 21, there is a high possibility that the nucleic acid probe 1243 is solidified in a sideways state on the fixing surface of the fixing part 1241, that is, the polymer layer 1242a, due to the electrical attraction.

The nucleic acid probe 1243 immobilized in a sideways state is no longer able to fully exhibit its original functions such as capturing target molecules.

According to the configuration of the present disclosure, as shown in FIG. 11, the nucleic acid probe 243 has an electrically neutral or positively charged functional group R added to at least some of the plurality of phosphate esters. ing. As a result, the charge of the nucleic acid probe 243 in the aqueous solution is changed to be electrically neutral or positively charged compared to before the functional group R is added. Therefore, when the nucleic acid probe 243 is immobilized on the fixing part 241, which has a positive electrical property, there is a possibility that the nucleic acid probe 243 will be solidified on the fixing surface of the fixing part 241, that is, on the polymer layer 242a, while lying on its side. can be reduced.

In addition, in the above configuration, the nucleic acid probes 243 whose charges are more electrically neutral than before the addition of the functional group R, are different from those of the adjacent nucleic acid probes 243 due to the charge of each of the nucleic acid probes 243. The repulsion between them is weak. Therefore, the nucleic acid probe 243 to which the functional group R is added can be immobilized on the immobilization part 241 at high density.

Furthermore, when the target molecule is a nucleic acid, the nucleic acid probe 243 having the above configuration has a high ability to specifically capture the target molecule because the repulsive force generated between the target molecule and the nucleic acid probe 243 is weak.

<Detection system 100>
Next, the detection system 100 using the nucleic acid probe 243 according to this embodiment will be explained. Here, only the points different from the detection system 100 according to the first to fourth embodiments described above will be explained. In addition to nucleic acids, the detection system 100 may detect peptides capable of binding to nucleic acids, transcriptional regulators, nucleic acid binding proteins such as polymerases, and the like. The detection system 100 includes, as an example, a detection device 3 and a cartridge 2 (flow path device) (see FIG. 2).

(Configuration of sensor unit 23)
FIG. 12 is a schematic diagram schematically showing an example of the internal configuration of the sensor unit 23 according to this embodiment. Here, only the points different from the sensor unit 23 described above will be explained.

As an example, the sensor unit 23 may include two sensors: a capture section 24 (sensor section) and a reference section 25 (sensor section). Below, when the sensor unit 23 has a single sensor, the sensor unit 23 may be simply referred to as a biosensor. When the sensor unit 23 has a plurality of sensors, for example, a capturing section 24 and a reference section 25, the capturing section 24 and the reference section 25 are collectively referred to as a biosensor if there is no need to distinguish between the individual sensors. Good too. Further, the entire sensor unit 23 including the capturing section 24 and the reference section 25 may be referred to as a biosensor.

The capture unit 24 has a nucleic acid probe 243 capable of capturing nucleic acids in the reaction solution on its surface, and captures the nucleic acids in the reaction solution. In this disclosure, the "surface" is intended to be the surface on which the nucleic acid probe 243 is placed. Details of the capture unit 24 will be described later.

The sensor unit 23 according to one embodiment is, for example, a sensor using elastic waves, and includes a capturing section 24, a reference section 25, a pair of first IDT (Inter Digital Transducer) electrodes 26A, a pair of second IDT electrodes 26B, and A substrate 27 is provided. The capture section 24, the reference section 25, the pair of first IDT electrodes 26A, and the pair of second IDT electrodes 26B may be located on the substrate 27.

The capturing section 24 and the reference section 25 of the sensor unit 23 may be sensors that utilize elastic waves, QCM (Quartz Crystal Microbalance), SPR (Surface Plasmon Resonance), FET (Field Effect Transistor), or the like, for example.

Furthermore, as another example, the sensor unit 23 may include a reaction section. The reaction section may include an amplification section and the above-mentioned capture section 24. The amplification section amplifies a nucleic acid having a predetermined sequence in the reaction solution. The reaction section is a place where the amplification reaction by the amplification section and the capture of the nucleic acid in the reaction solution by the capture section 24 are performed. In the reaction section, areas where amplification reactions and capture are performed may be divided. Furthermore, the amplification section where the amplification reaction proceeds does not need to be inside the sensor unit 23. Any location where a reaction solution is accommodated can serve as an amplification section. For example, when the amplification reaction is performed at a predetermined location in the channel 28, the predetermined location may be used as the amplification section.

FIG. 13 is a schematic diagram schematically showing an example of the configuration of the capturing section 24. The capture section 24 includes a fixing section 241, a polymer 242, and a nucleic acid probe 243. The fixing portion 241 and the polymer 242 are bonded at a bonding portion B1. Further, the polymer 242 and the nucleic acid probe 243 are bonded at a bonding portion B2. The fixing part 241 may be made of metal, for example. Specifically, the fixing portion 241 may be made of metals such as gold, chromium, and titanium, or a combination of these metals. The fixing portion 241 may be a single-layer metal film made of a single material, or may be a multi-layer metal film made of a plurality of materials. The material constituting the fixing part 241 is not limited to the metals mentioned above, and any material can be used as appropriate. The fixing part 241 may be manufactured by a conventionally known method.

In this embodiment, an example in which the fixing part 241 is made of gold (Au) will be described. As shown in bonding portion B1 in FIG. 13, the fixing portion 241 and the polymer 242 are bonded by a gold thiol bond (Au—S—). Furthermore, as shown in bonding portion B2 in FIG. 13, the polymer 242 and the nucleic acid probe 243 are bonded through an amide bond (-CONH-).

In this embodiment, the polymer 242 is 2-carboxy-N,N-dimethyl-N-[2'-(methacryloyl)oxyethyl]ethanaminium (3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate). ) (p-CBMA2) will be described.

Other examples of the polymer 242 include carboxybetaine methacrylate (CBMA) polymers other than CBMA2, sulfobetaine methacrylate (SBMA) polymers, and the like.

The nucleic acid probe 243 has a base sequence that binds to the target molecule to be captured. At least some of the plurality of phosphoric acid esters constituting the nucleic acid probe 243 have an electrically neutral functional group or a positively charged functional group added thereto.

The target molecule to be captured by the nucleic acid probe 243 is not particularly limited as long as it has the property of binding to the nucleic acid probe. For example, the target molecule may be a nucleic acid having a complementary base sequence to the base sequence of the nucleic acid probe. Alternatively, the target molecule may be a nucleic acid binding protein, including peptides and antibodies capable of binding to nucleic acids, transcriptional regulators, polymerases, and the like.

The pair of first IDT electrodes 26A may be arranged on the substrate 27 so as to sandwich the capture portion 24 therebetween. In the sensor unit 23 according to one embodiment, an electric signal (input signal) is input to one of the pair of first IDT electrodes 26A under the control of the detection device 3. The input electrical signal is converted into an elastic wave that propagates toward the capturing section 24 and is emitted from one first IDT electrode 26A. The emitted elastic waves pass through the trapping section 24 . The other first IDT electrode 26A can receive the elastic wave that has passed through the capture section 24.

In the capture unit 24, the target molecule to be captured interacts with the nucleic acid probe 243, thereby changing the propagation characteristics of the elastic wave propagating on the substrate 27. Specifically, the reaction between the target molecule and the nucleic acid probe 243 changes the weight applied to the substrate 27 or the viscosity of the liquid in contact with the surface of the substrate 27. The magnitude of these changes correlates with the amount of reaction between the target molecule and the nucleic acid probe 243.

Unlike the capture section 24, the nucleic acid probe 243 is not immobilized on the reference section 25. Therefore, in the reference portion 25, no reaction occurs between the target molecule and the nucleic acid probe 243. Therefore, the reference section 25 can function as a control for the capture section 24. The reference section 25 may be configured the same as or similar to the capture section 24 .

<Method for manufacturing sensor unit 23>
Next, a method for manufacturing the sensor unit (biosensor) 23 in this embodiment will be described. Here, in particular, a method for manufacturing the capturing section 24 included in the sensor unit 23 will be described using FIG. 14. FIG. 14 is a diagram illustrating an example of a method for manufacturing the trapping section 24 in this embodiment.

First, the nucleic acid probe 243 is manufactured including a preparation step S21 and a modification step S22.

First, a nucleic acid fragment having a base sequence that binds to a target molecule to be captured is prepared (S21: preparation step).

Next, at least some of the plurality of phosphate esters included in the nucleic acid fragment prepared in S21 are added with an electrically neutral functional group or a positively charged functional group. (S22; modification step).

Next, the fixing part 241 and the polymer 242 are combined (S23).

Next, the end of the polymer 242 bonded to the fixing part 241 in S23 is activated (S24).

Next, the nucleic acid fragment (nucleic acid probe) modified in S22 and the polymer 242 whose terminal was activated in S24 are bonded (S25: immobilization step).

The process of S25 can also be expressed as follows. The nucleic acid probe 243 manufactured by the method for manufacturing the nucleic acid probe 243 is immobilized on the immobilization part 241 in which the polymer 242 having positive electrical properties is disposed by activating the terminal.

Next, the ends of the polymer 242 that did not bind to the nucleic acid fragment in S25 are inactivated (S26), and the process ends.

Hereinafter, details of each step of the method for manufacturing the above-mentioned trapping part 24 will be explained using FIGS. 15 to 20.

(S21: Example of preparing a nucleic acid fragment)
In S21, a nucleic acid fragment having a base sequence that binds to a target molecule to be captured is prepared. The nucleic acid fragment can be prepared by synthesizing it by a conventionally well-known method. For example, nucleic acid fragments may be prepared by the phosphoramidite method.

(S22: Example of addition of functional group to phosphate ester included in nucleic acid fragment)
Next, an example of addition of a functional group to a phosphate ester included in a nucleic acid fragment in S22 will be described using FIG. 15. FIG. 15 is a diagram showing an example of addition of a functional group to a phosphate ester included in a nucleic acid fragment. An electrically neutral functional group R or a positively charged functional group R is added to at least some of the plurality of phosphate esters constituting the nucleic acid fragment. In the present disclosure, a nucleic acid fragment in which the functional group R is added to at least a portion of a phosphoric acid ester is used as the nucleic acid probe 243.

Furthermore, the functional group R added to the phosphoric ester may be at least one of an alkyl group, a methoxy group, an ethoxy group, a hydroxyl group, an aldehyde group, a ketone group, an amino group, and a mercapto group.

According to the above configuration, the charge of the nucleic acid probe 243 can be changed to electrically neutral or positive charge.

Further, the functional group added to the phosphoric acid ester may be a different type of functional group. For example, an alkyl group may be added to one phosphoric ester, and a functional group other than the alkyl group may be added to another phosphoric ester.

Furthermore, the functional group may be added to at least a portion of the phosphoric ester via a spacer.

Examples of spacers include polyethylene glycol (PEG). For example, PEG3 may be used as the spacer. Examples of other spacers include alkyl, polypropylene glycol, polybutylene glycol, and the like.

Further, as shown in FIG. 15, one functional group or two functional groups may be added to the phosphate ester at the 5' end of the nucleic acid fragment. The two functional groups added to the phosphoric acid ester may be different types of functional group R and functional group R1, or may be the same type of functional group R.

Further, NH ₂ may be added to the end of the nucleic acid probe 243 in order to form an amide bond with the polymer 242 whose end is esterified with NHS. Furthermore, NH ₂ may be added via a spacer.

(S23: Example of bonding between fixing part 241 and polymer 242)
Here, an example of the bonding between the fixing part 241 and the polymer 242 in S23 will be described using FIG. 16. FIG. 16 is a diagram showing an example of the connection between the fixing part 241 and the polymer 242. As shown in FIG. 16, in this example, p-CBMA2, which is the polymer 242, and Au, which is the fixing part 241, are bonded by a gold-thiol bond.

For example, the bond may be formed by a chemical reaction shown in the following formula.

In the above formula, the fixing part 241 is Au and the polymer 242 to be fixed is R-SH. "SH" is a thiol group, and "R" represents a portion of p-CBMA2 other than the thiol group.

Furthermore, as another example of a chemical reaction, the bond may be formed by a chemical reaction shown in the following formula.

In the above formula, the fixing part 241 is Au ₂ O ₃ and the polymer 242 to be fixed is R-SH. "SH" is a thiol group, and "R" represents a portion of p-CBMA2 other than the thiol group.

(S24: Example of terminal activation of polymer 242)
Next, an example of activation of the terminal of the polymer 242 in S24 will be explained using FIGS. 17 and 18. FIG. 17 is a diagram showing an example of activation of the terminal of a polymer. As shown in FIG. 17, EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) are reacted with p-CBMA2, which is the polymer 242 bonded to the fixing part 241. let This reaction converts COO ^- at the end of the side chain of polymer 242 into an NHS ester.

FIG. 18 is a diagram showing the change in charge of the polymer layer 242a due to activation of the terminal of the polymer 242. As shown in FIG. 18, before the terminal end of the polymer 242 is activated, the polymer layer 242a is entirely neutral because it is a zwitterionic polymer layer. As described above, activation of the end of polymer 242 converts the COO 2 ⁻ at the side chain end of polymer 242 to an NHS ester. This conversion changes the electrical properties of polymer 242 to a positive charge. As a result, after the terminal end of the polymer 242 is activated, the polymer layer 242a becomes positively charged.

In this example, the activation of the terminal of the polymer 242 is necessary for the formation of an amide bond between the nucleic acid probe 243 and the polymer 242 in S25.

(S25: Example of binding between nucleic acid probe 243 and polymer 242)
Next, an example of the binding between the nucleic acid probe (nucleic acid fragment) 243 and the polymer 242 in S25 will be described using FIG. 19. FIG. 19 is a diagram showing an example of binding between the nucleic acid probe 243 and the polymer 242. As shown in FIG. 19, for example, the end of the polymer 242 converted to NHS ester and the end of the nucleic acid probe 243 to which NH ₂ has been added may be bonded with an amide bond.

Furthermore, the bond between the nucleic acid probe 243 and the polymer 242 may be performed while the polymer layer 242a is positively charged, and is not limited to the above-mentioned amide bond.

Through this bonding, the nucleic acid probe 243 is immobilized on the immobilization part 241 on which the polymer 242 having a positive electrical property is disposed. That is, as shown in FIGS. 12 and 13, the nucleic acid probe 243 is immobilized on the immobilization part 241 of the capture part 24.

(S26: Example of terminal inactivation of polymer 242)
Next, an example of inactivation of the end of the polymer 242 that did not bind to the nucleic acid probe 243 in S26 will be described using FIG. 20. FIG. 20 is a diagram showing an example of inactivation of the end of the polymer 242 that did not bind to the nucleic acid probe 243. For example, the terminal NHS ester of the polymer 242 that is not bound to the nucleic acid probe 243 is inactivated by hydrolysis. For example, as shown in FIG. 20, by placing the polymer 242 in an alkaline environment, the end of the side chain of the polymer 242 having an NHS ester that is not bound to the nucleic acid probe 243 is returned to the COO ^{2 −} state before activation.

〔summary〕
A detection system 100 according to aspect 1 of the present disclosure includes an amplification unit 51 that performs an amplification reaction of a nucleic acid in a reaction solution that amplifies a nucleic acid having a predetermined sequence, and a first amplification unit that is capable of capturing the nucleic acid in the reaction solution. a reaction unit 50 having a capture unit 24 with a capture substance disposed on its surface; a detection device 3 capable of detecting a change in physical properties of the surface due to interaction between the nucleic acid and the first capture substance; Equipped with.

In the detection system 100 according to Aspect 2 of the present disclosure, in Aspect 1, the amplifying section 51 may have a surface on which the first capturing substance is disposed and functioning as the capturing section 24.

In the detection system 100 according to aspect 3 of the present disclosure, in

aspect

1 or 2, in the reaction section 50, the amplification reaction and the capture of the nucleic acid in the reaction solution by the first capture substance are performed simultaneously. Good too.

In the detection system 100 according to Aspect 4 of the present disclosure, in any of Aspects 1 to 3, the first capture substance is at least one of a nucleic acid serving as a template in the amplification reaction and a nucleic acid of an amplification product from the amplification reaction. It may be possible to capture either of them.

In the detection system 100 according to Aspect 5 of the present disclosure, in Aspect 4, the first capture substance is a first nucleic acid fragment F1 having a first complementary sequence complementary to the base sequence of the first end of the amplification product. , F11.

In the detection system 100 according to Aspect 6 of the present disclosure, in either Aspect 4 or 5, the first capture substance is a second complementary substance that is complementary to the base sequence at the second end of the nucleic acid serving as the template. The length of the second nucleic acid fragments F2 and F21, which contain the second nucleic acid fragments F2 and F21 having the sequence and capture the nucleic acid serving as the template, may be extended by the amplification reaction using the captured nucleic acid as a template. .

The detection system 100 according to Aspect 7 of the present disclosure is configured such that, in any of Aspects 1 to 6, the first capture substance is the third end of the nucleic acid strand that is elongated by the amplification reaction and is located on the side far from the surface. It may further contain a third nucleic acid fragment F3, F31 having a third complementary sequence complementary to the base sequence of the third nucleic acid fragment.

The detection system 100 according to aspect 8 of the present disclosure includes a translation unit 52 in which a translation reaction from the nucleic acid is performed in a reaction solution containing a nucleic acid having a predetermined sequence and ribosomes, and a translation unit 52 capable of capturing translation products in the reaction solution. a reaction section 50C having a capturing section 24C having a second capturing substance disposed on its surface; A device 3 is provided.

In the detection system 100 according to Aspect 9 of the present disclosure, in Aspect 8, the reaction solution may contain transfer RNA and amino acid molecules.

In the detection system 100 according to Aspect 10 of the present disclosure, in any of Aspects 1 to 9, the physical property may be at least one of surface acoustic waves, frequency, refractive index, and electrical conductivity.

In a detection system 100 according to an eleventh aspect of the present disclosure, in any one of the first to tenth aspects, the reaction section is a SAW (Surface Acoustic Wave), a QCM (Quartz Crystal Microbalance), an SPR (Surface Plasmon Resonance), or a FET. (Field Effect Transistor).

The detection system 100 according to aspect 12 of the present disclosure may further include a temperature adjustment section 37 that can adjust the temperature of the

reaction sections

50, 50A to 50C in any of the aspects 1 to 11.

The cartridge 2 according to aspect 13 of the present disclosure includes

reaction sections

50, 50A, and 50B that accommodate a reaction solution for amplifying a nucleic acid having a predetermined sequence, and an amplification section that performs an amplification reaction of the nucleic acid in the reaction solution. 51, 51A, 51B, and a

reaction section

50, 50A, 50B having a

capture section

24, 24A, 24B on the surface of which a first capture substance capable of capturing the nucleic acid in the reaction solution; an attachment portion 29 for attachment to a detection device capable of detecting changes in the physical properties of the surface due to interaction with a first capture substance.

The cartridge 2 according to aspect 14 of the present disclosure is a reaction section 50 that accommodates a liquid containing a target nucleic acid having a predetermined sequence, the reaction unit 50 having a capture substance disposed on the surface that can capture the target nucleic acid in the liquid. a reaction section 50 having a reaction section 24; and an attachment section 29 for attachment to a detection device capable of detecting a change in the physical properties of the surface due to the interaction between the target nucleic acid and the capture substance, The substance includes a first capture region containing first nucleic acid fragments F1 and F11 having a first complementary sequence complementary to the base sequence of the first end of the target nucleic acid, and the first end of the target nucleic acid. has a second capture region including second nucleic acid fragments F2 and F21 having a second complementary sequence complementary to a different base sequence at the second end, and the capture section 24 includes SAW (Surface Acoustic Wave), The sensor uses one of QCM (Quartz Crystal Microbalance), SPR (Surface Plasmon Resonance), or FET (Field Effect Transistor).

The cartridge 2 according to aspect 15 of the present disclosure includes a reaction section 50C that accommodates a reaction solution containing a nucleic acid having a predetermined sequence and a ribosome, and a translation section 52 in which a translation reaction from the nucleic acid is performed in the reaction solution. and a trapping portion 24C having a second trapping substance on its surface capable of trapping the translation product in the reaction solution, and an interaction between the translation product and the second trapping substance. and an attachment part 29 for attachment to a detection device capable of detecting changes in the physical properties of the surface.

A detection method according to aspect 16 of the present disclosure includes: an amplification unit 51 that performs an amplification reaction of a nucleic acid in a reaction solution that amplifies a nucleic acid having a predetermined sequence; and a first capture unit that can capture the nucleic acid in the reaction solution. a containment step of accommodating the reaction solution in a reaction section 50 having a capture section 24 with a substance disposed on its surface; an amplification step of carrying out an amplification reaction of the nucleic acid in the reaction solution accommodated in the reaction section; , a detection step of detecting a change in physical properties of the surface due to the interaction between the nucleic acid and the first capture substance.

The detection method according to aspect 17 of the present disclosure includes a translation unit 52 in which a translation reaction from the nucleic acid is performed in a reaction solution containing a nucleic acid having a predetermined sequence and a ribosome, and a translation unit 52 capable of capturing a translation product in the reaction solution. a containing step of accommodating the reaction solution in a reaction section 50C having a capture section 24C having a second capture substance disposed on its surface; and a step of accommodating the reaction solution from the nucleic acid in the reaction solution accommodated in the reaction section 50C. and a detection step of detecting a change in physical properties of the surface due to the interaction between the translation product and the second capture substance.

In the detection system according to aspect 18 of the present disclosure, in the aspect 1 or 8, the capturing section has a nucleic acid probe, the nucleic acid probe has a base sequence that binds to a target molecule to be captured, and An electrically neutral functional group or a positively charged functional group is added to at least some of the plurality of phosphate esters constituting the nucleic acid probe.

In the detection system according to Aspect 19 of the present disclosure, in Aspect 18, the functional group is at least one of an alkyl group, a methoxy group, an ethoxy group, a hydroxyl group, an aldehyde group, a ketone group, an amino group, and a mercapto group. It may be.

In the detection system according to Aspect 20 of the present disclosure, in Aspect 18 or 19, the electrically neutral functional group or the positively charged functional group is attached to one of the phosphoric esters via a spacer. It may be added to at least a part of it.

In the detection system according to aspect 21 of the present disclosure, in aspects 18 to 20, the nucleic acid probe may be electrically neutral or positively charged in an aqueous solution with a pH in the range of pH 6 to pH 8. .

In the detection system according to aspect 22 of the present disclosure, in the aspect 1 or 8, the capturing section has a nucleic acid probe, and the nucleic acid probe is a nucleic acid probe having a base sequence that binds to a target molecule to be captured. A cation may be coordinated to at least a portion of the plurality of phosphate esters constituting the nucleic acid probe.

A detection system according to Aspect 23 of the present disclosure is a detection system according to any one of Aspects 18 to 22, in which a sensor part in which the nucleic acid probe is immobilized on a fixing part on which a substance having a positive electrical property is disposed is provided. You may prepare.

A method for producing a nucleic acid probe according to aspect 24 of the present disclosure includes a preparation step of preparing a nucleic acid fragment having a base sequence that binds to a target molecule to be captured; and a modification step of modifying at least a portion of the material by adding an electrically neutral functional group or a positively charged functional group.

In the method for producing a nucleic acid probe according to Aspect 25 of the present disclosure, in Aspect 24, the target molecule is a nucleic acid, and the nucleic acid fragment prepared in the preparation step has a base sequence that the nucleic acid that is the target molecule has. may have a complementary base sequence.

A method for producing a biosensor according to Aspect 26 of the present disclosure includes a method for producing a biosensor, in which a nucleic acid probe produced by the method for producing a nucleic acid probe according to

Aspect

24 or 25 is fixed to a fixing part on which a substance having positive electrical properties is disposed. It includes an immobilization step of immobilizing on.

The invention according to the present disclosure has been described above based on the drawings and examples. However, the invention according to the present disclosure is not limited to each embodiment described above. That is, the invention according to the present disclosure can be modified in various ways within the scope shown in the present disclosure, and the invention according to the present disclosure also applies to embodiments obtained by appropriately combining technical means disclosed in different embodiments. Included in technical scope. In other words, it should be noted that those skilled in the art can easily make various changes or modifications based on the present disclosure. It should also be noted that these variations or modifications are included within the scope of this disclosure.

2 Cartridge 3 Detection device 23 Sensor unit (biosensor)
24, 24A to 24C Capture section (sensor section, biosensor)
25 Reference part (sensor part, biosensor)
29 Attachment section 37

Temperature adjustment section

50, 50A to

50C Reaction section

51, 51A, 51B Amplification section 52 Translation section 100 Detection system 241 Fixation section 243 Nucleic acid probe F1, F11 First nucleic acid fragment F2, F21 Second nucleic acid fragment F3, F31 Third nucleic acid fragment R, R1 Functional group S1 Preparation step S2 Modification step

Claims

It has an amplification section that performs an amplification reaction of the nucleic acid in a reaction solution that amplifies the nucleic acid having a predetermined sequence, and a capture section that has a first capture substance on its surface that can capture the nucleic acid in the reaction solution. a reaction part;
a detection device capable of detecting a change in the physical properties of the surface due to the interaction between the nucleic acid and the first capture substance;
A detection system comprising:
The detection system according to claim 1, wherein the amplification section has a surface on which the first capture substance is disposed and functions as the capture section.
In the reaction section, the amplification reaction and the capture of the nucleic acid in the reaction solution by the first capture substance are performed simultaneously.
The detection system according to claim 1 or 2.
4. The first capturing substance is capable of capturing at least one of a nucleic acid serving as a template in the amplification reaction and a nucleic acid of an amplification product from the amplification reaction. detection system.
The detection system according to claim 4, wherein the first capture substance includes a first nucleic acid fragment having a first complementary sequence that is complementary to the base sequence of the first end of the amplification product.
The first capture substance includes a second nucleic acid fragment having a second complementary sequence that is complementary to the base sequence at the second end of the template nucleic acid, and the second nucleic acid fragment captures the template nucleic acid. The length of is extended by the amplification reaction using the captured nucleic acid as a template.
The detection system according to claim 4 or 5.
The first capture substance further includes a third nucleic acid fragment having a third complementary sequence that is complementary to the base sequence at the third end of the nucleic acid chain extended by the amplification reaction, which is located on the side far from the surface. ,
The detection system according to any one of claims 1 to 6.
a translation part in which a translation reaction from the nucleic acid is carried out in a reaction solution containing a nucleic acid having a predetermined sequence and ribosome; a capture part having a second capture substance on its surface capable of capturing translation products in the reaction solution; a reaction section having;
a detection device capable of detecting a change in the physical properties of the surface due to the interaction between the translation product and the second capture substance;
A detection system comprising:
The reaction solution contains transfer RNA and amino acid molecules,
9. Detection system according to claim 8.
The detection system according to any one of claims 1 to 9, wherein the physical property is at least one of surface acoustic waves, frequency, refractive index, and electrical conductivity.
Claims 1 to 10, wherein the reaction section is a sensor that uses any one of SAW (Surface Acoustic Wave), QCM (Quartz Crystal Microbalance), SPR (Surface Plasmon Resonance), or FET (Field Effect Transistor). The detection system according to any one of the above.
further comprising a temperature adjustment section that can adjust the temperature of the reaction section;
12. A detection system according to any one of claims 1 to 11.
A reaction section that contains a reaction solution for amplifying a nucleic acid having a predetermined sequence, an amplification section that performs an amplification reaction of the nucleic acid in the reaction solution, and a first capture that can capture the nucleic acid in the reaction solution. a reaction part having a trapping part with a substance disposed on its surface;
an attachment part for attaching to a detection device capable of detecting a change in the physical property of the surface due to the interaction between the nucleic acid and the first capture substance;
Cartridge with.
a reaction part containing a liquid containing a target nucleic acid having a predetermined sequence, the reaction part having a capture part on the surface of which a capture substance capable of capturing the target nucleic acid in the liquid;
an attachment part for attachment to a detection device capable of detecting a change in the physical properties of the surface due to the interaction between the target nucleic acid and the capture substance;
The capture substance has a first capture region including a first nucleic acid fragment having a first complementary sequence complementary to the base sequence of the first end of the target nucleic acid, and the first end of the target nucleic acid. a second capture region comprising a second nucleic acid fragment having a second complementary sequence complementary to a different base sequence at the second end;
The capturing unit is a sensor that uses one of SAW (Surface Acoustic Wave), QCM (Quartz Crystal Microbalance), SPR (Surface Plasmon Resonance), or FET (Field Effect Transistor).
cartridge.
A reaction part containing a reaction solution containing a nucleic acid having a predetermined sequence and a ribosome, a translation part in which a translation reaction from the nucleic acid is performed in the reaction solution, and a translation part capable of capturing translation products in the reaction solution. a reaction section having a capture section with a second capture substance disposed on its surface;
an attachment for attachment to a detection device capable of detecting changes in physical properties of the surface due to interaction of the translation product and the second capture substance;
Cartridge with.
It has an amplification section that performs an amplification reaction of the nucleic acid in a reaction solution that amplifies the nucleic acid having a predetermined sequence, and a capture section that has a first capture substance on its surface that can capture the nucleic acid in the reaction solution. a containing step of containing the reaction solution in a reaction section;
an amplification step of performing an amplification reaction of the nucleic acid in the reaction solution contained in the reaction section;
a detection step of detecting a change in physical properties of the surface due to the interaction between the nucleic acid and the first capture substance;
Detection methods including.
a translation part in which a translation reaction from the nucleic acid is carried out in a reaction solution containing a nucleic acid having a predetermined sequence and ribosome; a capture part having a second capture substance on its surface capable of capturing translation products in the reaction solution; accommodating the reaction solution in a reaction section having;
a translation step of performing a translation reaction from the nucleic acid in the reaction solution contained in the reaction section;
a detection step of detecting a change in physical properties of the surface due to the interaction of the translation product and the second capture substance;
Detection methods including.
The capturing section has a nucleic acid probe,
The nucleic acid probe is a nucleic acid probe having a base sequence that binds to a target molecule to be captured,
Claim 1 or Claim 8, wherein an electrically neutral functional group or a positively charged functional group is added to at least some of the plurality of phosphoric acid esters constituting the nucleic acid probe. Detection system described in .
The functional group is at least one of an alkyl group, a methoxy group, an ethoxy group, a hydroxyl group, an aldehyde group, a hydroxyl group, an aldehyde group, a ketone group, an amino group, and a mercapto group.
19. Detection system according to claim 18.
The electrically neutral functional group or the positively charged functional group is added to at least a portion of the phosphoric acid ester via a spacer.
20. Detection system according to claim 18 or 19.
The detection system according to any one of claims 18 to 20, wherein the nucleic acid probe is electrically neutral or positively charged in an aqueous solution in a pH range of 6 to 8.
The capturing section has a nucleic acid probe,
The nucleic acid probe is a nucleic acid probe having a base sequence that binds to a target molecule to be captured,
A cation is coordinated to at least a portion of the plurality of phosphate esters constituting the nucleic acid probe.
The detection system according to claim 1 or claim 8.
a sensor part in which the nucleic acid probe is immobilized on a fixing part in which a substance having a positive electrical property is disposed;
23. Detection system according to any one of claims 18 to 22.
a preparation step of preparing a nucleic acid fragment having a base sequence that binds to a target molecule to be captured;
a modification step of modifying at least some of the plurality of phosphate esters included in the nucleic acid fragment by adding an electrically neutral functional group or a positively charged functional group; include,
Method for producing nucleic acid probes.
the target molecule is a nucleic acid;
The nucleic acid fragment prepared in the preparation step has a base sequence complementary to the base sequence of the nucleic acid that is the target molecule.
The method for producing a nucleic acid probe according to claim 24.
An immobilization step of immobilizing the nucleic acid probe produced by the method for producing a nucleic acid probe according to claim 24 or 25 on an immobilization part in which a substance having a positive electric charge is disposed.
Biosensor manufacturing method.