JP2003299485A - Temperature control-type microreactor and microreactor system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a temperature control-type microreactor highly compatible with automatic analysis, enabling a nucleic acid to be detected in high accuracy in a short time using a small amount of a sample and also applicable to the aimed nucleic acid aliquot and its amplification by PCR technique, and to provide a microreactor system using said microreactor. <P>SOLUTION: The temperature control-type microreactor has flow channels whose surface is immobilized with a deoxyribonucleic acid or ribonucleic acid, wherein the temperature of the flow channels can be controlled at positions where the deoxyribonucleic acid or ribonucleic acid is immobilized. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention has a high adaptability to automatic analysis and can detect a nucleic acid with high accuracy in a short time using a small amount of sample. The present invention relates to a temperature-controlled microreactor applicable to amplification and a microreactor system using the same.

[0002]

2. Description of the Related Art Deoxyribonucleic acid or ribonucleic acid (nucleotide probe, hereinafter also referred to as probe) having an artificially designed base sequence is used as a means for analyzing a sample containing various kinds of nucleic acids such as deoxyribonucleic acid or ribonucleic acid.
There is known a method of performing detection by binding to a specific nucleic acid in a sample with a complementary strand (hybridization) by using.

This method can also be applied to detect many kinds of nucleic acid at once by using an extremely large number of probes, for example, tens of thousands kinds or more. Such a method is described in Science vol. 270, 467-470
(1995). However, since the complementary strand bond between nucleic acids is a result of the formation of many hydrogen bonds between the probe and the base of the sample nucleic acid, the strength of the bond is greatly affected by the base sequence constituting the probe,
The temperature at which hybridization is possible differs depending on the base sequence that constitutes the probe.

That is, although complementary strand bonds between nucleic acids are made by hydrogen bonds between bases, the bond between adenine (A) and thymine (T) or the bond between adenine (A) and uracil (U) is 2 per base. While there are hydrogen bonds at one place, the bond between guanine (G) and cytosine (C) is 3 per base.
It is a hydrogen bond at one place. Therefore, the thermal stability of complementary strand binding becomes higher as the base sequence constituting the probe is rich in guanine (cytosine), and becomes lower as it is rich in adenine (thymine or uracil). The thermal stability of a complementary strand bond is generally represented by a temperature (melting temperature: Tm) at which 50% binding and 50% dissociation occur, but hybridization occurs at a temperature near the Tm of the complementary strand bond of each probe. It is preferable to carry out. Complementary strand bond is difficult to be formed even if hybridization is performed under a temperature higher than Tm,
On the contrary, when hybridization is carried out at a temperature lower than Tm, mismatch binding is likely to occur.

Therefore, for example, assuming that the Tm of the complementary strand bond of the A probe is 60 ° C. and the Tm of the complementary strand bond of the B probe is 40 ° C., if the hybridization temperature is set to 60 ° C., it corresponds to the A probe. A nucleic acid having a sequence corresponding to the B probe can be detected, but a nucleic acid having a sequence corresponding to the B probe cannot be detected. However, if the hybridization temperature is set to 40 ° C., for example, a nucleic acid having a sequence corresponding to the B probe can be detected, but false detection of the nucleic acid corresponding to the A probe will increase. Thus, setting the hybridization temperature is very important.

On the other hand, Japanese Patent Laid-Open No. 2001-23546
No. 9 discloses a method of improving the detection accuracy by changing the hybridization temperature for each fixed position of the probe. However, in order to bring the entire surface of the probe into contact with the sample by this method, it was necessary to use a large amount of sample and to make the contact time between the sample and the probe long, for example, one day and night. In order to secure a large amount of sample, it is necessary to increase the sample amount of cells, microorganisms, etc., or to amplify a small amount of sample by the polymerase chain reaction method (PCR method). However, it is often difficult in terms of cost and time to obtain a large amount of valuable experimental materials such as cells and microorganisms. Further, in the PCR method, the amplification effect may differ depending on the base sequence of the nucleic acid, or a base sequence different from the original sequence (mutated) may be amplified. In addition, a long contact time inevitably results in a reduction in work efficiency, and is not suitable when a large number of sample treatments are required per time such as genetic diagnosis. Due to the above problems, the method disclosed in JP 2001-235469 A is also insufficient.

[0007]

In view of the above situation, the present invention has high applicability to automatic analysis, is capable of detecting nucleic acid with high accuracy in a short time using a small amount of sample, and It is an object of the present invention to provide a temperature control type microreactor applicable to fractionation of cells and amplification by PCR method and a microreactor system using the same.

[0008]

DISCLOSURE OF THE INVENTION The present invention provides a microreactor or a μTAS (micro / m) which has been remarkably studied in recent years.
iniaturized Total Amarysi
As a result of intensive studies focusing on the technology of S. System), the accuracy of nucleic acid detection and a number of useful methods were improved by incorporating the concept of controlling the temperature at each probe fixing location in a flow injection type μTAS device. The inventors have found that it has an effect and completed the present invention. The present invention is a temperature-controlled microreactor having a channel on which deoxyribonucleic acid or ribonucleic acid is immobilized, which can control the temperature of the channel at the position where the deoxyribonucleic acid or ribonucleic acid is immobilized. Is a temperature-controlled microreactor. The present invention is described in detail below.

The temperature-controlled microreactor of the present invention has deoxyribonucleic acid (hereinafter also referred to as DNA) on the surface.
Alternatively, it has a channel in which ribonucleic acid (hereinafter, also referred to as RNA) is immobilized. By performing the hybridization in the channel, even a trace amount of sample can be reliably brought into contact with the immobilized DNA or RNA (hereinafter, also referred to as probe) in the process of passing through the channel. In addition, the washing step, which is essential for improving the sensitivity in detecting nucleic acids by hybridization, can be performed reliably and easily. Further, the step of hybridizing the sample and the step of washing the inside of the reactor can be performed in the same apparatus, which facilitates automation.

The probe is not particularly limited as long as it has a base sequence corresponding to the target nucleic acid, but when the target nucleic acid is fractionated using the temperature-controlled microreactor of the present invention, It preferably contains a base sequence that can be digested with a restriction enzyme. As a result, if a restriction enzyme is used, only specific nucleic acids can be easily separated. The fractionated nucleic acid can be used for other analyzes such as cloning and sequencing, further increasing the value of the reactor according to the present invention.

The DNA or RNA used as a probe in the present invention is not only those obtained from nature such as microorganisms, animals (humans, etc.), plants and viruses, but also those synthesized by a DNA synthesizer and various modifications (biotinylation). , Thiolation, methylation, addition of a fluorescent dye, etc.). In particular, DNA or R containing at least partially a base sequence of a human gene or a base sequence complementary thereto
When NA is used as a probe, the temperature-controlled microreactor of the present invention analyzes human gene sequences, for example, sequence, gene diagnosis, single nucleotide substitution mutation polymorphism (SNP).
s) It can be used for analysis. When the sample contains 16S ribosomal RNA derived from a microorganism, the microorganism can be identified by immobilizing DNA or RNA containing at least partially a base sequence complementary thereto.

When performing simultaneous analysis of many kinds of DNA or RNA, it is preferable that the immobilized DNA or RNA has two or more kinds of base sequences. This allows efficient analysis of many kinds of nucleic acids at the same time.

The method for immobilizing DNA or RNA on the surface of the channel of the temperature-controlled microreactor of the present invention is not particularly limited, and a conventionally known technique may be used. For example, in the case where the flow path is made of glass, various surface treatment agents are used to introduce amino groups, aldehyde groups, or the like on the glass surface to immobilize the DNA or RNA 5 ′ or 3 ′.
A method of introducing functional groups or molecules / atomic groups such as amino groups, aldehyde groups, thiol groups, and biotin into the ends to covalently bond the two, or treating the glass surface with a cation such as polylysine to make DNA or RNA negative. Examples thereof include a method of electrostatically coupling with an electric charge. Among them, the method of immobilizing by covalent bond is preferable because peeling of the immobilized nucleic acid is suppressed.

As a process for immobilizing particularly many kinds of DNA or RNA, a technique used in so-called DNA chip (DNA microarray or oligo DNA microarray) can be exemplified. DNA
The microarray is prepared by a method in which a solution containing probe DNA prepared separately in advance is arranged on a chip surface in a minute volume of a few nL to a few pL by a special device such as a spotter or an arrayer and fixed to a specific area on a substrate. In this case, a probe DNA having an amino group, an aldehyde group, a thiol group, biotin or the like introduced as a functional group at the terminal, and a substrate surface-treated with various silane coupling agents having an amino group, an aldehyde group, an epoxy group or the like are used. Thus, the probe DNA can be immobilized by covalent bond. Also,
The oligo DNA microarray is produced by a method of directly synthesizing probe DNA on a substrate. As an example, there is a method of selectively synthesizing DNA in a predetermined region of a minute matrix by using a substance having a protective group that is selectively removed by light irradiation and combining photolithography technology and solid-phase synthesis technology. . In the present invention, the above-mentioned spotter, photolithography technique, or the like may be used for the base material in which the flow path is formed by the flow path forming method described below. When the irregularities of the base material due to the formation of the flow channel become a problem, DNA or the like may be immobilized on the base material having no irregularity, and then bonded to the base material having the flow channel formed thereon. As described above, according to the present invention, a microreactor on which an extremely large number of types of DNA or RNA are immobilized can be easily prepared.

The concentration for immobilizing the above DNA or RNA is not particularly limited, but the preferred lower limit is 0.001 pmol / since the immobilization state can be easily confirmed.
mm ² . The area on which the DNA or RNA is immobilized is not particularly limited, but it is preferable to immobilize the DNA or RNA so as to surround the periphery in the traveling direction of the solution in order to improve the detection sensitivity.

The temperature-controlled microreactor of the present invention can actively control the temperature of the flow channel at the position where DNA or RNA is immobilized. This makes it possible to quickly set the optimum hybridization temperature for the base sequence of each probe, greatly improve the nucleic acid detection accuracy, and shorten the reaction time.

The temperature controllable range is 0 to 10
0.0 ° C is preferred. When the temperature range is narrower than this range, for example, when the control range is 40 to 60 ° C., the detection sensitivity of the probe having the optimum hybridization temperature out of the range becomes poor, and it becomes difficult to collect the bound sample. It may become.

When the temperature is raised to a high temperature such as 95 ° C. within the above temperature control range, bubbles may be generated in the reactor. The generated air bubbles disturb the uniform flow of the sample and hinder the contact between the sample and the immobilized DNA or RNA, which is not preferable. The method for suppressing the generation of bubbles is not particularly limited, and examples thereof include a method of degassing the sample solution before flowing it into the reactor and a method of increasing the internal pressure of the reactor while flowing the sample solution. Examples of the degassing method include known methods such as vacuum degassing, ultrasonic degassing, and heat degassing. Examples of the method of increasing the internal pressure of the reactor include a method of using a reactor having a structure in which a valve is installed in the middle of a flow path.

The temperature control accuracy is preferably within ± 0.5 ° C. with respect to the set value when the temperature control type microreactor is used. Outside this range, it is difficult to obtain the effects of the present invention. More preferably within ± 0.1 ° C. Also, sorting out a sequence similar to a certain base sequence, etc.
Even when a plurality of types of nucleic acids are hybridized with one probe, the highly accurate temperature control as described above is effective.

The heating and / or cooling means for controlling the temperature is not particularly limited, and examples thereof include a method of using a heater, a heating element, an air cooling fan, a cooling element, a thermoelectric conversion element or the like alone or in combination. . Above all,
A thermoelectric conversion element containing a thermoelectric conversion material having a thermoelectric effect,
Since the heating and cooling can be performed by adjusting the direction of the current, the hybridization temperature can be controlled precisely and quickly, which is preferable. Further, by using the thermoelectric conversion element, it is possible to simplify and downsize the entire device. As such a thermoelectric conversion element, for example,
P-type or N-type thermoelectric conversion element in which an appropriate dopant is added to an alloy containing at least two or more elements selected from the group consisting of Bi, Te, Se and Sb elements, and a so-called Peltier element in which they are modularized Is mentioned. Further, as a method for producing these thermoelectric conversion elements, for example, Bi, Te, Se or Sb powder and a predetermined amount of a dopant and a dopant are weighed, the powder is mixed and melted, and the obtained alloy lump is crushed to obtain an alloy powder. Then, a method of sintering and the like can be mentioned.

The means for measuring the temperature for controlling the temperature is not particularly limited. For example, a thermistor,
Examples thereof include a method using a temperature detecting element such as a thermocouple and a platinum temperature measuring element. Among them, the method using a thermistor is preferable in terms of strictness of temperature control.

However, since the thermoelectric conversion element and the temperature detecting element are expensive, it is preferable to install these elements so as to come into contact with the flow path through the outer surface of the temperature control type reactor. As a result, the reactor portion can be made disposable and the thermoelectric conversion element and the temperature detection element can be reused.

When the DNA or RNA to be immobilized is composed of two or more kinds of base sequences and each of them is fractionated or analyzed, it is preferable that the immobilization site is divided into two or more sites and each can be controlled at different temperatures. By setting the optimum hybridization temperature for different kinds of probes, it is possible to analyze many kinds of nucleic acids with high accuracy.

When the molecular weights of the nucleic acids in the sample do not vary widely, it is preferable to arrange the probes so that the hybridization temperature becomes higher to lower in order from the upstream of the channel. When heat denaturing the sample nucleic acid and hybridizing it, the upstream side of the flow path is at a higher temperature, so when the sample is flowed through the microreactor, the highest upstream nucleic acid is the highest at the immobilized position. Suitable hybridization can be carried out at temperature. Then, suitable hybridization can be carried out at a temperature lower than the downstream of the previous step. Conversely, if a probe with a low hybridization temperature is immobilized upstream of a probe with a high hybridization temperature, it is necessary to re-denature the nucleic acid in the sample, or false detection is performed at the immobilization position of the probe with a low hybridization temperature. May occur.

Further, in the temperature-controlled microreactor of the present invention, it is preferable that the temperature can be controlled by time division and / or area division when controlling the temperature at a large number of positions in the channel. When temperature control is performed for a large number of nucleic acid-immobilized positions on the flow channel, it is uneconomical in terms of cost to prepare a control system at each position. Therefore, heating and / or
Alternatively, a cooling means and a temperature detection element are prepared and controlled by one temperature control device in a time-sharing manner, or heating and / or cooling means is prepared at each of a plurality of positions, and one temperature detection element is provided. It is preferable that the temperature control is performed by area division by one temperature control device.

FIG. 1 is a schematic view showing one embodiment of the temperature-controlled microreactor of the present invention. The base material 5 is not particularly limited, and inorganic materials such as glass and ceramics;
Organic compounds containing plastics such as polymethylmethacrylate and polydimethylsiloxane; those composed of metals and the like. Among them, those made of glass, thermoplastics or metal are preferable in consideration of recyclability, and those made of plastics are more preferable in view of processability. The element for adjusting the temperature is manufactured by using a known technique, and the substrate 5 is used.
It may be fixed on or formed directly on the substrate, or fixed or formed on another substrate as shown in FIG.

A channel 4 for flowing a sample is formed on the base material 5. The sectional area of the flow path 4 is not particularly limited, but is preferably 1 mm ² or less. If it exceeds 1 mm ² , temperature control of the flow channel may become difficult. More preferably, it is 0.09 mm ² or less.

The method of forming the flow path 4 on the base material 5 depends on the material of the base material 5, the size of the flow path, and the required processing accuracy, but for example, laser processing or a photosensitive glass material is used. Examples of the technique include exposure through a photomask, photolithography and etching, atomic beam etching, molding techniques such as injection molding generally used in plastic processing, and lamination of base materials in which flow path shapes are formed in advance. With these techniques, a three-dimensional flow channel structure as well as a flat surface can be formed.

In the present invention, the flow path is formed mainly by the fine processing technique as described above. Therefore, the contact area of the immobilized DNA or RNA with respect to the sample can be made very large, and the analysis ability per unit area is extremely high. Further, the temperature control, which is a feature of the present invention, can be performed easily and extremely accurately. Furthermore, since the shape of the flow path that can be formed is not limited, the analysis work can be speeded up and simplified by combining with other reactors, and a part of the sample can be collected by branching in the middle of the flow path. It is also possible to perform other analysis work in.

The substrate having the flow channel formed thereon should be bonded to another substrate if a means for moving the sample in the flow channel, for example, pressure feeding of a liquid using a syringe pump or the like is required to seal the flow channel. Is preferred.

The size of the temperature control type microreactor of the present invention is not particularly limited, but it is preferably 1000 cm ³ or less in consideration of handling property as a microreactor.

As the sample to be subjected to the temperature-controlled microreactor of the present invention, DNA obtainable from nature such as microorganisms, animals (humans, etc.), plants, viruses, etc.
Alternatively, a solution or droplet containing not only RNA but also one synthesized by a DNA synthesizer and one obtained by subjecting DNA or RNA to various modifications (biotinylation, thiolation, methylation, addition of a fluorescent dye, etc.) can be mentioned. For solutions, etc., Southern blotting
Inorganic salts such as SSC solution (citric acid-sodium chloride aqueous solution) commonly used in, a surfactant such as sodium dodecyl sulfate, and an organic compound such as polyethylene glycol may be contained. With these, the accuracy of hybridization can be adjusted. When a human-derived nucleic acid or a nucleic acid having the same base sequence as that of a human is used as a sample, the temperature-controlled microreactor of the present invention is used for analysis of human gene sequences, for example, for gene diagnosis and 1-base substitution mutation polymorphism (SNPs) analysis. be able to. In addition, when the sample contains 16S ribosomal RNA derived from a microorganism, the microorganism can be identified by immobilizing DNA or RNA complementary thereto.

However, when the sample nucleic acid is not single-stranded such as double-stranded or triple-stranded, the sample nucleic acid may be denatured into single-stranded by heat denaturation or the like before being subjected to the test. It is preferable for improving the detection sensitivity.

As described above, the temperature-controlled microreactor of the present invention is used for nucleic acid analysis of each individual / species of microorganisms, viruses, etc. existing in the environment such as soil sphere, hydrosphere, or biota such as microbial groups. It can be used for nucleic acid analysis for the whole, and nucleic acid analysis for each solid / biological species related to animals including humans, plants and the like.

As one embodiment of the temperature-controlled microreactor of the present invention, a method for fractionating nucleic acid using the temperature-controlled microreactor of the present invention will be described with reference to FIG. This temperature-controlled microreactor has 7, 8,
9 probes having three different base sequences are immobilized. The base sequence of each probe is the restriction enzyme E.
Can be digested in one place with coRI. 8 in this example
It is assumed that a nucleic acid having the nucleotide sequence of is desired to be collected.

In the operation, first, the sample is flown into this temperature-controlled microreactor, and the immobilized nucleic acid is hybridized with the nucleic acid in the sample. After washing, the temperature of the temperature-controlled microreactor was set to 4
Lower to ℃. Next, the reactor is filled with a solution containing the restriction enzyme EcoRI and substances necessary for the enzymatic reaction. At this time, the liquid temperature of the reactor is maintained at 4 ° C. Then, only the temperature at the position of the probe having the base sequence of 8 is raised to 37 ° C. at which the restriction enzyme acts. 37 ° C. is the temperature that is optimal for the restriction enzyme while the hybridization is not lost. Therefore, restriction enzyme digestion occurs at this position. On the other hand, in other parts of the flow path, the temperature is too low and digestion with the restriction enzyme does not occur. After a certain period of time, the temperature of the position of the probe having the base sequence of 8 was changed to 4 ° C.
Then, the liquid in the reactor is collected. At this time, the nucleic acid is detached from the substrate only at the position where digestion occurs,
Only specific nucleic acids can be recovered.

Further, as an improvement of the above-mentioned method for fractionating nucleic acids, there is a method of designing a nucleotide sequence so that adjacent probes digest with different restriction enzymes during immobilization. As a result, it is possible to prevent digestion from occurring at an unexpected position of the worker. Another example of the method for fractionating nucleic acids is a method in which probes digestible with different restriction enzymes are immobilized.

Next, as one embodiment of the temperature-controlled microreactor of the present invention, the temperature-controlled microreactor of the present invention is used to amplify and isolate a specific nucleic acid sequence using the polymerase chain reaction method (PCR method). A method for taking the same will be described with reference to FIG. Probes having four different base sequences of 10, 11, 12, and 13 are immobilized on the temperature-controlled microreactor.
Then, the base sequence of each probe can be digested at one site with the restriction enzyme EcoRI. And 10, 11, 1
It is assumed that 3 probes are immobilized so that they do not overlap at all, whereas 11 and 12 probes are immobilized at the same position and target the same nucleic acid in the sample. However, the probe having 11 nucleotide sequences hybridizes to the (+) strand (or sense strand) of the sample nucleic acid, while the probe having 12 nucleotide sequences (-) strand (or antisense strand). And a probe having a nucleotide sequence of 11 and 12
The hybridization temperature is different from that of the probe having the nucleotide sequence of, and the hybridization temperature of the probe having the nucleotide sequence of 11 is higher. In this example, it is assumed that a sample that hybridizes with a probe having 11 nucleotide sequences is to be amplified and fractionated.

In the operation, first, the sample is flown into this temperature-controlled microreactor, and the nucleic acid in the sample is hybridized with the immobilized probe. At this time, the probes having the base sequences of 11 and 12 immobilized at the same position can hybridize only the probe having the base sequence of 11 if adjusted to the hybridization temperature of the probe having the base sequence of 11. it can. After washing, the temperature of the temperature-controlled microreactor is lowered to 4 ° C. Next, a temperature control type microreactor is filled with a solution containing a restriction enzyme EcoRI and a PCR enzyme, and substances necessary for carrying out an enzymatic reaction or a PCR reaction. At this time, the liquid temperature of the reactor is maintained at 4 ° C. Then, the temperature at the position where the probe having the 11 nucleotide sequence and the probe having the 12 nucleotide sequence is immobilized is raised to 37 ° C. at which the restriction enzyme acts. As a result, the immobilized probes having the nucleotide sequences of 11 and 12 are released from the substrate, and these are used as primers and templates in the PCR reaction. Then 11 and 12
The temperature at the position of the probe having the nucleotide sequence of is raised to a temperature at which the PCR reaction occurs. Elsewhere in the flow path, the temperature is too low for the PCR reaction to occur. After the reaction cycle of the PCR reaction is completed, the liquid temperature is returned to 4 ° C., and then the liquid in the reactor is collected. At this time, since the probe is detached from the substrate only at the position where the digestion occurs, it is possible to recover only the specific nucleic acid.

Further, as an improvement of the method of amplifying and fractionating a specific nucleic acid sequence using the polymerase chain reaction method (PCR method) using the above-mentioned temperature-controlled microreactor of the present invention, a specific nucleic acid in a sample There is also a method of designing a probe that hybridizes with a nucleic acid that serves as a primer for PCR reaction so that it can be digested with another restriction enzyme. This makes it possible to more strictly control the primer concentration in the PCR reaction.

A microreactor system characterized by using the temperature-controlled microreactor of the present invention is also one aspect of the present invention. The microreactor system of the present invention comprises the temperature-controlled microreactor of the present invention and peripheral equipment. Examples of the peripheral devices include a device for moving a sample in the temperature-controlled microreactor (hereinafter also referred to as a moving device), a device for collecting a liquid that has passed through the temperature-controlled microreactor (hereinafter also referred to as a collecting device), and a fixed device. An apparatus (hereinafter, also referred to as a detection apparatus) for detecting a sample hybridized with the converted probe can be used. The temperature-controlled microreactor of the present invention can be used in combination with another microreactor, for example, a PCR microreactor, or integrated on the same substrate.

Examples of the moving device include a syringe pump which has an advantage that pulsating flow does not occur, an air pump which can be easily brought into the field not only in the laboratory, but also as an electrode and a power source for performing electrophoresis for easy sample movement control. Is mentioned. Examples of the recovery device include an autosampler commonly used in liquid chromatography and the like. Examples of the detection device include a PCR thermal cycler, a surface plasmon resonance device, a thermal lens microscope, a mass spectrometer, an ultraviolet spectrophotometer, and a fluorescence scanner.

Further, the microreactor system of the present invention has an apparatus for thermally denaturing a sample consisting of double-stranded or triple-stranded nucleic acid into a single strand on the same substrate as or separately from the temperature-controlled microreactor. It is preferable. If this device is installed separately, preheat the sample,
Examples include a device that denatures a double strand or the like into a single strand and then rapidly cools it with ice water or the like. When this operation is performed on the same substrate as the temperature-controlled microreactor, a method of denaturing the inside of the channel at a position closer to the sample injection side than the position where the probe is immobilized can be mentioned. .

The denaturing temperature varies depending on the base sequence of the nucleic acid of the sample, but is preferably 90 ° C., more preferably 95 ° C. or higher. If the temperature is lower than 90 ° C., the denaturation of the nucleic acid of the sample will be insufficient, and the detection sensitivity will often be significantly reduced. However, when denaturing on the same substrate as the temperature-controlled microreactor, the heating that accompanies the denaturation causes the air etc. dissolved in the sample solution to vaporize in the reactor and inhibit hybridization. Sometimes. Therefore, it is preferable to degas the sample solution before heating. The degassing method is not particularly limited, and examples thereof include known methods such as vacuum degassing, ultrasonic degassing, and heat degassing.

By combining the above-mentioned devices, the microreactor of the present invention can be used not only indoors but also in a site where analysis is required.

By using the temperature-controlled microreactor and the microreactor system of the present invention, it is possible to perform analysis with higher sensitivity than before. This means that even those that could not be measured by the conventional method due to the problem of sample amount can be analyzed. Moreover, since the step of flowing the liquid through the specific flow path is the basic, automation is easy. This is useful for improving the efficiency and labor saving of analysis work.

In addition, for example, when the microbial species in the activated sludge of a sewage treatment plant is analyzed as an analysis target, it is expected that effects such as improvement of treatment capacity and prevention of bulking can be expected. Similarly, if it is used to analyze the dynamics of microorganisms in the soil at the bioremediation construction site, it is possible to effectively carry out the construction and shorten the period. This enhances the utility of bioremediation technology.

When used for gene diagnosis such as SNPs analysis of human genes, the test can be performed with less misdiagnosis, and the usefulness of diagnosis can be enhanced.

By using the temperature-controlled microreactor and the microreactor system of the present invention, not only analysis of nucleic acid but also specific nucleic acid can be fractionated.
Since the separated nucleic acid can be used for the analysis of Nya, this device is useful for the efficiency of research and development using the nucleic acid.

[0050]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

(Example 1) (1) DNA-immobilized cover glass (length 76 mm, width 26 mm, thickness 0.0)
Chloroform and 0.1 M hydrochloric acid were dripped in 8 mm).
Next, NH ₄ OH / H ₂ O ₂ / H ₂ O was added to the cover glass.
Drop the solution (weight ratio 1: 1: 5) and add HCl / H
_{A 2} O ₂ / H ₂ O solution (weight ratio 1: 1: 5) was dripped. Next, a 2% γ-aminopropyltriethoxysilane (hereinafter, also referred to as APTES) -toluene solution was dropped on this cover glass and left at room temperature for 24 hours. Next, a 1% maleic anhydride-chloroform solution was dripped onto this cover glass, and further 0.2MN-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride (hereinafter, EDC) and 0.05MN-hydroxy were added. The succinimide mixed solution was dripped. Next, a 1M ethanolamine hydrochloride aqueous solution (pH 9) was dropped on this cover glass. And then, 5'of array 1 on this cover glass
A synthetic DNA modified with a terminal thiol group was dropped. Finally, the synthetic DNA was immobilized by leaving it at room temperature for a whole day and night.

(2) Production of temperature-controlled microreactor Cover glass (length 76 mm, width 26 mm, thickness 0.0
8 mm) with another cover glass (length 76 mm, width 13 m)
m, thickness 0.30 mm) so that two sheets do not overlap, and a glass with a thickness of 0.15 mm is sandwiched between the two sheets and fixed with clips, and the silicone resin (Product name: SR
2410, manufactured by Toray Dow Corning Co., Ltd.) was dropped and adhered. After the bonding, the sandwiched glass was removed to obtain a rectangular parallelepiped cover glass having a concave cross section and one groove on the upper surface and a thickness of 0.38 mm. On this laminated cover glass,
A cover glass on which the synthetic DNA obtained in (1) was fixed was laminated, and this was also adhered with the same resin. As a result, the flow path was sealed and a reactor in which DNA was immobilized by covalent bond could be produced. The width of the channel is 0.15m in width.
m, the depth was 0.30 mm, and the flow path length was 76 mm.
The area where the immobilized DNA is exposed in the channel is 1.5.
It was mm ² .

(3) Setting of peripheral equipment Each equipment was connected as shown in FIG. As each device, a commercially available device was used.

(4) DNA detection experiment Using boiling distilled sterile water, hybridization buffer (6 × SSC, 0.2% SDS, 5 × De)
nhardt's solution, 50% formamide) was prepared and injected into a syringe. This syringe was set in the pump and flowed at a flow rate of 0.5 ml / hr. At the same time, use a DNA denaturation heater at 95 ° C (preferred denaturation temperature for the sample),
Immobilization unit heater at 60.5 ° C (sample and immobilized DN
A hybridization temperature suitable for A) was set to heat the channel.

Pause pump, array 500 ng 2
Sample buffer (6 x SSC,
0.2% SDS, 5 × Denhardt's solution, 50
3% of formamide) was injected into the pipe. afterwards,
The pump was turned on again. The dye contained in the sample is D
After passing through the NA fixing position, the buffer solution was flown for another 60 minutes. After that, set the heater of the immobilization unit to 95 ° C,
The eluate was collected in the liquid collecting section.

Using the collected sample as a template
A PCR reaction was carried out using the primers of Sequence 3 and Sequence 4. Then, it was detected by ordinary gel electrophoresis and ethidium bromide staining. The obtained gel electrophoresis image was visually observed in the dark under UV irradiation, and the detection intensity was evaluated according to the following criteria. ⊚: Detection by strong staining ◯: Detection by weak staining ×: No detection by staining The results are shown in Table 1.

Comparative Example 1 (1) Immobilization of DNA and D
Preparation of NA Chip By the same method as in Example 1, cover glass (length 26 mm, width 26 m) with thiol group-modified synthetic DNA of Sequence 1 was prepared.
m, thickness 0.08 mm). The fixed area was 1.5 mm ² . Next, a cover glass having a thickness of 0.3 was left around the area where the DNA was immobilized, leaving an area of 12 mm ² (3 × 4 mm, equivalent to the channel area of Example 1).
mm resin film.

(2) DNA detection experiment A sample buffer solution containing 6 ng of the DNA of sequence 2 (6
X SSC, 0.2% SDS, 5 x Denbardt's
3 μl of the solution, 50% formamide) was dropped on the immobilized DNA, and the upper part of the reactor was closed with another cover glass (a state in which the resin film was sandwiched between the cover glasses).
This was set on a Peltier device at 95 ° C. After heating for 10 seconds, it was cooled to 60.5 ° C. in 1 minute. Then, the cover glass on the top of the reactor was removed, and the liquid on the chip was removed. Then hybridization buffer (6 x SSC, 0.2% SDS, 5 x Denhard
A t's solution, 50% formamide) was added dropwise onto the immobilized DNA and then heated at 95 ° C for 1 minute to collect the solution. A PCR reaction was carried out using the recovered liquid as a template and the primers of Sequence 3 and Sequence 4, and detection was performed by ordinary gel electrophoresis and ethidium bromide staining. The obtained gel electrophoresis image was visually observed in the dark under the irradiation of ultraviolet rays, and the detection intensity was evaluated according to the same criteria as in Example 1.
The results are shown in Table 1.

[0059]

[Table 1]

(Example 2) (1) Immobilization of DNA and preparation of microreactor By the same method as in Example 1, 5'of Sequence 1 and Sequence 5 was prepared.
The terminal thiol group-modified synthetic DNA was immobilized on the reactor shown in FIG. Also, the production of the microreactor was performed in the same manner as in Example 1.

(2) Setting of peripheral equipment Each equipment was connected as shown in FIG. As each device, a commercially available device was used.

(3) DNA Detection Experiment Using boiling sterile distilled water, hybridization buffer (6 × SSC, 0.2% SDS, 5 × De)
nhardt's solution, 50% formamide) was prepared and injected into a syringe. This syringe was set in a syringe pump and flowed at a flow rate of 0.5 ml / hr. At the same time, the DNA denaturation heater is 95 ℃, Peltier element (high temperature side)
Was set to 61.0 ° C. and the Peltier element (low temperature side) was set to 60.7 ° C. to heat the flow path. At this time, the temperature of the Peltier element was controlled by each thermistor, and the channel temperature was measured by the channel temperature detecting element.

The pump was paused and 500 ng of DNA of sequence 2 and 6 respectively and sample buffer containing dye (6 × SSC, 0.2% SDS, 5 × Denhard).
3 μl of t's solution, 50% formamide) was injected into the pipe. Then the pump was turned on again. After the dye contained in the sample has passed through the DNA fixing position, another 60
The buffer was run for a minute. After that, the Peltier element on the low temperature side was first set to 95 ° C., and the hybridized DN
A was collected. Next, the Peltier element on the high temperature side was set to 95 ° C., and the hybridized DNA was recovered.

PCR using the recovered solution of Peltier on the low temperature side as a template and the primers of Sequence 3 and Sequence 4
The reaction was carried out. Separately, PCR reaction was performed using the primers of Sequence 7 and Sequence 8. A PCR reaction was carried out using the sequence 7 and sequence 8 primers with the recovered solution of the Peltier on the high temperature side as a template. Separately, PCR reaction was performed using the primers of Sequence 3 and Sequence 4. Each of these was detected by conventional gel electrophoresis and ethidium bromide staining. The obtained gel electrophoresis image was visually observed in the dark under UV irradiation, and the detection intensity was evaluated according to the following criteria. ◯: with detection x: without detection The results are shown in Table 2.

[0065]

[Table 2]

(Example 3) (1) Immobilization of DNA and preparation of microreactor By the same method as in Example 1, the thiol group-modified synthetic DNAs of Sequences 9 and 10 were immobilized in the reactor of FIG. Also, the production of the microreactor was performed in the same manner as in Example 1.

(2) Setting of peripheral equipment Each equipment was connected as shown in FIG. As each device, a commercially available device was used.

(3) DNA Detection Experiment Using boiling distilled sterile water, hybridization buffer (6 × SSC, 0.2% SDS, 5 × De)
nhardt's solution, 50% formamide) was prepared and injected into a syringe. This syringe was set in a syringe pump and flowed at a flow rate of 0.5 ml / hr. At the same time, the Peltier element for denaturing the DNA was set to 95 ° C. and the Peltier element (for the immobilization section) was set to 66.0 ° C., and the channel was heated. At this time, the temperature of the Peltier element was controlled by each thermistor, and the channel temperature was measured by the channel temperature detecting element.

The pump is temporarily stopped and the DNA of sequence 6 is added to 5
Sample buffer (6xSSC, containing 00 mg and dye,
0.2% SDS, 5 × Denhardt's solution, 50
3% of formamide) was injected into the pipe. afterwards,
The pump was turned on again.

After the dye contained in the sample passed through the DNA fixing position, the buffer was further flowed for 60 minutes. Next, the temperature of the flow path was set to 4 ° C., 10 units of restriction enzyme ApaLI (manufactured by Takara Shuzo), and enzyme for DNA amplification Takara T.
aq5 unit (Takara Shuzo) and amplification solution (10
mMTris-HCl (pH 8.2), 50 mMKC
1, 1.5M MgCl ₂ , 0.05 mM DTT (Dit
hiothreitol), 0.2 mM dATP,
0.2 mM dTTP, 0.2 mM dCTP, 0.2
The inside of the reactor was replaced with (mM dGTP).

After the replacement, the operation of the pump was stopped, the Peltier element of the immobilization section was set at 37 ° C., and the enzyme was reacted for 15 minutes. Then, the Peltier element of the immobilization section was operated for 20 cycles, with one cycle consisting of 95 ° C. for 1 minute, 61.0 ° C. for 2 minutes, and 72.0 ° C. for 3 minutes. Then, the first buffer was reflowed to recover the amplified DNA. The recovered DNA was detected by ordinary gel electrophoresis and staining with ethidium bromide. The obtained gel electrophoresis image was visually observed in the dark under UV irradiation, and the detection intensity was evaluated according to the following criteria. ◯: Detected ×: Not detected The results are shown in Table 3.

[0072]

[Table 3]

(Example 4) (1) Immobilization of DNA and preparation of microreactor By the same method as in Example 1, 5'of Sequence 1 and Sequence 5 was prepared.
The terminal thiol group-modified synthetic DNA was immobilized on the reactor shown in FIG. Also, the production of the microreactor was performed in the same manner as in Example 1. At this time, the switching device was installed as shown in FIG. This device is installed between a Peltier device and a thermistor and a temperature control device, and switches the Peltier device on the high temperature side and the low temperature side and switches the thermistor on the high temperature side and the low temperature side.

(2) Setting of peripheral equipment Each equipment was connected as shown in FIG. As each device, a commercially available device was used.

(3) DNA detection experiment Using boiling distilled sterile water, a hybridization buffer (6 × SSC, 0.2% SDS, 5 × De) was prepared.
nhardt's solution, 50% formamide) was prepared and injected into a syringe. This syringe was set in a pump and flowed at a flow rate of 0.1 ml / hr. DNA denaturation heater 95 ℃, Peltier element (high temperature side) 61.0 ℃,
The Peltier device (low temperature side) was set at 60.7 ° C. The switching device switches between the high temperature side and the low temperature side every one second.

The pump was temporarily stopped, and 500 ng each of the DNAs of sequence 2 and sequence 6 and a sample buffer containing dye (6 × SSC, 0.2% SDS, 5 × Denbard) were used.
3 μl of t's solution, 50% formamide) was injected into the pipe. Then the pump was turned on again.

After the dye contained in the sample passed through the DNA fixing position, the buffer solution was flown for another 60 minutes. afterwards,
First, the low temperature Peltier device was set at 95 ° C., and the hybridized DNA was recovered. Next, the Peltier element on the high temperature side was set to 95 ° C., and the hybridized DNA was recovered.

PCR was performed by using the primers of Sequences 3 and 4 with the recovered solution of Peltier on the low temperature side as a template.
The reaction was carried out. Separately, PCR reactions were carried out using the primers of Sequence 7 and Sequence 8. Also, using the recovered liquid of the Peltier on the high temperature side as a template,
And a PCR reaction was performed using the primers of Sequence 8. Separately, PCR reaction was performed using the primers of Sequence 3 and Sequence 4. Then, it was detected by ordinary gel electrophoresis and ethidium bromide staining.
The obtained gel electrophoresis image was visually observed in the dark under UV irradiation, and the detection intensity was evaluated according to the following criteria. ◯: with detection x: without detection The results are shown in Table 4.

[0079]

[Table 4]

[0080]

EFFECTS OF THE INVENTION According to the present invention, nucleic acids can be detected with high accuracy in a short period of time with a high degree of adaptability to automatic analysis and using a small amount of sample. A temperature-controlled microreactor applicable to amplification and a microreactor system using the same can be provided.

[0081]

[Sequence list] <110> SEKISUI CHEMICAL CO., LTD. <120> Temperature controlled microreactor and microreactor system <130> 00P00507 <160> 10 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 tcggtgatgt cggcgatata 20 <210> 2 <211> 200 <212> DNA <213> Artificial Sequence <400> 2 ttc tcg gag cac tgt ccg acc gct ttg gcc gcc gcc cag tcc tgc tcg 48 ctt cgc tac ttg gag cca cta tcg act acg cga tca tgg cga cca cac 96 ccg cgc ggg atc gag atc tcg atc ctc tac gcc gga cgc atc gtg gcc 144 ggc atc acc ggc gcc aca ggt gcg gtt gct ggc gcc tat atc gcc gac 192 atc acc ga 200 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 tcggtgatgt cggcgatata 20 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <400> 4 ttctcggagc actgtccg 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 cggcacctgt cctacgagtt 20 <210> 6 <211> 200 <212> DNA <213> Artificial Sequence <400> 6 ttg ttt cgg cgt ggg tat ggt ggc agg ccc cgt ggc cgg ggg act gtt 48 ggg cgc cat ctc ctt gca tgc acc att cct tgc ggc ggc ggt gct caa 96 cgg cct tca acc cag tca gct cct tcc ggt ggg cgc ggg gca tga cta 144 tcg tcg ccg cac tta tga ctg tct tct tta tca tgc aac tcg tag gac 192 agg tgc cg 200 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 cggcacctgt cctacgagtt 20 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <400> 8 ttgtttcggc gtgggtat 18 <210> 9 <211> 46 <212> DNA <213> Artificial Sequence <400> 9 cgtttcggtg atgacggtga gtgcaccggc acctgtccta cgagtt 46 <210> 10 <211> 44 <212> DNA <213> Artificial Sequence <400> 10 cgtttcggtg atgacggtga gtgcacttgt ttcggcgtgg gtat 44

[Brief description of drawings]

FIG. 1 is a schematic view showing an embodiment of a temperature-controlled microreactor of the present invention.

FIG. 2 is a schematic view showing an embodiment of a temperature-controlled microreactor of the present invention in which an element for adjusting temperature is fixed or formed on another substrate.

FIG. 3 is a schematic diagram showing an embodiment of the temperature-controlled microreactor of the present invention in which probes having three different base sequences are immobilized and nucleic acids can be collected.

FIG. 4 is a schematic diagram showing one embodiment of the temperature-controlled microreactor of the present invention in which probes having four different base sequences are immobilized and which is capable of carrying out a PCR method.

5 is a schematic diagram showing the microreactor system used in Example 1. FIG.

FIG. 6 is a schematic diagram showing a microreactor used in Example 2.

FIG. 7 is a schematic diagram showing a microreactor system used in Example 2.

FIG. 8 is a schematic diagram showing a microreactor used in Example 3.

FIG. 9 is a schematic diagram showing a microreactor system used in Example 3.

FIG. 10 is a schematic diagram showing a microreactor used in Example 4.

FIG. 11 is a schematic diagram showing a microreactor system used in Example 4.

[Explanation of symbols]

1 Heating and / or cooling elements 2 Temperature detection element 3 Immobilized nucleic acid 4 channels 5 base materials 6 substrate 7 Immobilized nucleic acid 8 Immobilized nucleic acid 9 Immobilized nucleic acid 10 Immobilized nucleic acid 11 Immobilized nucleic acid 12 Immobilized nucleic acid 13 Immobilized nucleic acid

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 35/08 G01N 37/00 101 37/00 101 C12N 15/00 ZNAA // G01N 27/447 G01N 27 / 26 315Z

Claims (7)

[Claims] 1. A temperature-controlled microreactor having a channel on which deoxyribonucleic acid or ribonucleic acid is immobilized, the temperature of the channel being controllable at the position where the deoxyribonucleic acid or ribonucleic acid is immobilized. A temperature-controlled microreactor characterized by being a thing. 2. The temperature-controlled microreactor according to claim 1, wherein the deoxyribonucleic acid or ribonucleic acid has two or more kinds of base sequences. 3. The temperature-controlled microreactor according to claim 1, wherein the deoxyribonucleic acid or ribonucleic acid contains a base sequence that can be digested with a restriction enzyme. 4. The temperature-controlled microreactor according to claim 1, 2 or 3, which can be controlled at two or more locations in the flow path at different temperatures. 5. The temperature of the flow path can be controlled by time division and / or area division.
The temperature-controlled microreactor according to 2, 3, or 4. 6. A temperature-controlled microreactor according to claim 1, wherein a thermoelectric conversion material is used for heating and / or cooling. 7. A microreactor system comprising the microreactor according to claim 1, 2, 3, 4, 5 or 6.