JP2008134199A - Microfluid-device electrophoretic method and system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for separating double-stranded nucleic acids under the presence of denaturing gradient using a microfluid device on the basis of the differences of base sequences by a double-stranded nucleic acid electrophoretic method, and to provide a system for the method. <P>SOLUTION: The method includes the process of forming denaturing gradient by moving a solution from a microchannel for liquid introduction, the process of introducing double-stranded nucleic acids from a microchannel for sample introduction to a microchannel for separation, the process of separating the introduced double-stranded nucleic acids, and the process of detecting the separated double-stranded nucleic acids. The temperature of a part at which the microchannel for liquid introduction is positioned is controlled during the forming process of the denaturing gradient in the method and the system suitable for the method. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for separating a double-stranded nucleic acid based on a difference in base sequence by electrophoresis using a microfluidic device, and an apparatus for the method. Furthermore, the present invention relates to a method for separating double-stranded nucleic acids based on differences in base sequences by double-stranded nucleic acid electrophoresis in the presence of a denaturing agent concentration gradient using a microfluidic device, and an apparatus for the method.

  The following prior arts are known for separation techniques using capillaries or microfluidic devices. Japanese Patent Publication No. 2001-515204 (Patent Document 1) discloses a microfluidic system for determining the temperature of a fluid, which includes a sensor operably connected to a channel and a temperature-responsive energy source. Is disclosed. Japanese Patent Application Laid-Open No. 2003-117409 (Patent Document 2) discloses a microchemical analysis device characterized in that a microchip is molded using a heat-resistant plastic and provided with temperature adjusting means. ing. Japanese Unexamined Patent Application Publication No. 2004-279340 (Patent Document 3) discloses a microchip having a plurality of temperature sensors. International Publication No. WO2002 / 090912 (Patent Document 4) discloses a microchip microflow characterized in that a liquid phase temperature in a microchannel on a microchip is measured in a non-contact manner by detecting light emission intensity from a fluorescent substance. A method for measuring the temperature of the liquid phase in the channel is disclosed. However, these prior arts are not related to separation techniques based on differences in the base sequences of double-stranded nucleic acids.

  Japanese Patent Application Laid-Open No. 10-502738 (Patent Document 5) is known as one relating to a technique for separating a double-stranded nucleic acid in a capillary. This relates to a method for separating double-stranded nucleic acid by non-micellar electrophoresis in an electrophoretic separation medium, and utilizes the fact that the denaturation temperature differs due to the difference in DNA base sequence. That is, this separation technique uses a temperature change over time as a condition for weakening the hydrogen bond between the double strands.

By the way, it is known that double-stranded nucleic acid is separated by electrophoresis using a microfluidic device (Patent Document 6). The features of the apparatus using the microfluidic device are as follows.
1. An apparatus for separating double-stranded DNA based on a difference in base sequence, a denaturant concentration gradient forming part for forming a DNA denaturant concentration gradient, a sample introducing part for introducing a DNA sample into the apparatus, and denaturation A microchip electrophoresis apparatus including an electrophoresis unit that has a denaturant concentration gradient region formed by an agent concentration gradient forming unit and electrophoretically separates a DNA sample introduced by a sample introduction unit.
2. The denaturant concentration gradient forming section includes a first reservoir filled with a denaturant-containing buffer, a second reservoir filled with a buffer, a first channel communicating with the first reservoir, A second channel communicating with the second reservoir, a third channel connecting the first channel and the second channel, and the first reservoir includes a first electrode and a first electrode. And a second reservoir including a second electrode and a second power source connected to the second electrode, wherein the first power source and the second power source are connected to each other. Using the electroosmotic flow generated by controlling the voltage of the denaturant, the denaturant-containing buffer solution and the buffer solution are mixed at an arbitrary ratio to form a denaturant concentration gradient region in the third channel, and the denaturant concentration Introducing the gradient region into the electrophoresis section. The 1, butterflies. Microchip electrophoresis device.
3. The denaturant concentration gradient forming section includes a first reservoir filled with a denaturant-containing buffer, a second reservoir filled with a buffer, a first channel communicating with the first reservoir, A second channel communicating with the second reservoir, a third channel connecting the first channel and the second channel, and the first reservoir includes a first electrode and a first electrode. And a first pump connected to the second reservoir, the second reservoir including a second electrode and a second power source connected to the second electrode; and A second pump is connected to control the voltage of the first power source and the second power source and the flow rate of the first pump and the second pump, so that the denaturant-containing buffer solution and the buffer solution can be added at an arbitrary ratio. Mix to form a denaturant gradient region in the third channel And a denaturant concentration gradient region is introduced into the electrophoresis section. Microchip electrophoresis device.
4). The sample introduction section includes a third reservoir filled with a DNA sample, a fourth reservoir for a DNA sample waste solution, a fourth channel communicating with the third reservoir and the third channel, 4 reservoir and a fifth channel communicating with the third channel, and the third reservoir further includes a third electrode and a third power source connected to the third electrode, Furthermore, the fourth reservoir includes a fourth electrode and a fourth power source connected to the fourth electrode, and the electroosmotic flow generated by controlling the voltage of the third power source and the fourth power source. And a DNA sample is introduced into the third channel using electrophoresis. Microchip electrophoresis device.
5. The electrophoresis unit includes a fifth reservoir for waste liquid and the third channel communicating with the fifth reservoir. The fifth reservoir further includes a fifth electrode and a fifth electrode. A fifth power source connected thereto; and 3. the denaturant concentration gradient forming part described above; A microchip electrophoretic separation comprising a DNA sample introduced into the third channel in a denaturant concentration gradient region formed in the third channel, including the sample introduction part described above Electrophoresis device.
6). The electrophoresis unit includes a fifth reservoir for waste liquid and the third channel communicating with the fifth reservoir, and the fifth reservoir includes a fifth electrode and a fifth electrode. A fifth power source connected thereto; and 3. the denaturant concentration gradient forming part described above; A microchip electrophoretic separation comprising a DNA sample introduced into the third channel in a denaturant concentration gradient region formed in the third channel, including the sample introduction part described above Electrophoresis device.
Special table 2001-515204 gazette JP 2003-117409 A JP 2004-279340 A International Publication No. 2002/090912 Pamphlet Japanese National Patent Publication No. 10-502738 International Publication No. 2005/049196 Pamphlet

  However, the apparatus and analysis method using the microfluidic device described in International Publication No. 2005/049196 are not satisfactory from the viewpoint of efficiency and the like, and a new method and apparatus therefor are required. ing. For example, since the viscosity of the denaturant-containing solution introduced into the microchannel is high, it can be considered that introduction into the microchannel is very difficult. Therefore, there is a possibility that a device such as a high-performance pump is required, or a microfluidic device that can withstand a high-pressure fluid is required. In addition, when the material of the inner wall of the microchannel is a material such as an acrylic resin that does not easily cause electroosmotic flow, there is a problem that it takes a very long time to introduce the solution when using electroosmotic flow. there were. These problems may not be able to achieve the high speed, space saving, low running cost, etc. that are advantages of the analysis using the microfluidic device.

  Therefore, the present invention provides a double-stranded nucleic acid electrophoretic method using a microfluidic device in the presence of a denaturing agent concentration gradient. The object is to provide a method of separation. It is another object of the present invention to provide a microfluidic system suitable for the method.

  In view of such a situation, the present inventors have completed the present invention as a result of intensive studies.

That is, the present invention
First,
Microchannel for separation,
Two liquid introduction microchannels connected to the separation microchannel, and a sample introduction microchannel connected to the separation microchannel;
, By performing electrophoresis in the presence of a denaturant concentration gradient using a microfluidic device having at least a double-stranded nucleic acid based on a difference in base sequence,
The method
at least,
(A) A first liquid containing a denaturant is introduced into one microchannel of the two liquid introduction microchannels, and at the same time, the concentration is lower than the concentration of the denaturant of the first liquid into the other microchannel. Introducing a second liquid containing or not containing a denaturant to form a denaturant concentration gradient in the separation microchannel,
(B) introducing a double-stranded nucleic acid from the sample introduction microchannel into the separation microchannel;
(C) separating the introduced double-stranded nucleic acid, and (d) detecting the separated double-stranded nucleic acid,
Including
Controlling the temperature of the liquid-introducing microchannel during the step (a),
Second, microchannel for separation,
Two liquid introduction microchannels connected to the separation microchannel, and a sample introduction microchannel connected to the separation microchannel;
, By performing electrophoresis in the presence of a denaturant concentration gradient using a microfluidic device having at least a double-stranded nucleic acid based on a difference in base sequence,
The method
at least,
(A) A first liquid containing a denaturant is introduced into one microchannel of the two liquid introduction microchannels, and at the same time, the concentration is lower than the concentration of the denaturant of the first liquid into the other microchannel. Introducing a second liquid containing a denaturant or not containing a denaturant to form a denaturant concentration gradient in the separation microchannel,
(B) introducing a double-stranded nucleic acid from the sample introduction microchannel into the separation microchannel;
(C) separating the introduced double-stranded nucleic acid, and (d) detecting the separated double-stranded nucleic acid,
Including
The method, wherein the temperature of the portion where the liquid-introducing microchannel is located is set to 30 to 80 ° C. during the step (a),
Third,
Microchannel for separation,
Two liquid introduction microchannels connected to the separation microchannel, and a sample introduction microchannel connected to the separation microchannel;
, By performing electrophoresis in the presence of a denaturant concentration gradient using a microfluidic device having at least a double-stranded nucleic acid based on a difference in base sequence,
The method
at least,
(A) A first liquid containing a denaturant is introduced into one microchannel of the two liquid introduction microchannels, and at the same time, the concentration is lower than the concentration of the denaturant of the first liquid into the other microchannel. Introducing a second liquid containing a denaturant or not containing a denaturant to form a denaturant concentration gradient in the separation microchannel,
(B) introducing a double-stranded nucleic acid from the sample introduction microchannel into the separation microchannel;
(C) separating the introduced double-stranded nucleic acid, and (d) detecting the separated double-stranded nucleic acid,
Including
During the step (a), the temperature of the portion where one of the microchannels for liquid introduction is located is raised, and the temperature of the portion where the other microchannel is located is lowered at the same time, Said method,
Fourth,
The method according to the second aspect, wherein after the step (a), the temperature of the portion where the liquid introduction microchannel is located is 10 to 30 ° C.,
Fifth,
5. The method according to the second or fourth method, wherein a temperature of a portion where the liquid introduction microchannel is located is set to 10 to 30 ° C. during the steps (b) to (c).

Sixth,
The method according to the second or third method, characterized in that, during the step (b), the temperature of the portion where the sample introduction microchannel is located is 10 to 30 ° C.,
Seventh,
During the step (d), the temperature of at least a part of the part where the separation microchannel is located and the part where the separated double-stranded nucleic acid is present is 10 to 50 ° C. The method according to the second or third,
Eighth,
During the step (a), the temperature of the portion where one microchannel of the liquid introduction microchannel is located is increased, and the temperature of the portion where the other microchannel is located is lowered at the same time. The method according to the third aspect, wherein the amount of change in temperature of the microchannel for use is 30 ° C to 80 ° C,
Ninthly, during the step (a), the temperature of the portion where one of the liquid introduction microchannels is located is set so that the viscosity of the liquid introduced into the one microchannel is one third to two. The temperature is raised to a fraction, and at the same time, the temperature of the portion where the other microchannel is located is lowered so that the viscosity of the liquid introduced into the other microchannel is 2 to 3 times. The method according to the third aspect,
Tenth, microchannel for separation,
Two liquid introduction microchannels connected to the separation microchannel,
A sample introduction microchannel connected to the separation microchannel,
At least one temperature control means for controlling the temperature of at least a part of the portion where the separation microchannel is located;
At least one temperature control means for controlling the temperature of at least a part of the portion where the two liquid introduction microchannels are located, and controls the temperature of at least a part of the portion where the sample introduction microchannel is located At least one temperature control means,
A microfluidic system for separating double-stranded nucleic acids based on differences in base sequences by performing electrophoresis in the presence of a denaturant concentration gradient,
It is.

  According to the method of the present invention, a double-stranded nucleic acid electrophoresis method using a microfluidic device in the presence of a concentration gradient of a denaturant can efficiently separate a double-stranded nucleic acid by a difference in base sequence and with a good resolution. it can. In addition, the method of the present invention makes it possible to form a denaturant concentration gradient with good reproducibility and efficiency, so that separation of double-stranded nucleic acids can be performed with good reproducibility and efficiency.

  In addition, the microfluidic system of the present invention can perform separation of double-stranded nucleic acid efficiently and with good separation ability.

  The method and apparatus of the present invention will be described with reference to FIGS.

  The microfluidic device used in the method of the present invention comprises at least a separation microchannel (3), two liquid introduction microchannels (1) and (2) connected to the separation microchannel (3), and A sample introduction microchannel (4) connected to the separation microchannel (3). Such microchannels can be produced by using well-known techniques such as photolithography technology, precision molding using a mold, laser processing, precision machining, and other suitable materials (glass, quartz, plastic, silicon, etc.). Resin).

  The groove width, groove depth and length of each channel (1) to (4) can be arbitrarily selected. The groove width and depth are 1 μm to 1000 μm, preferably 10 μm to 500 μm, and more preferably 30 μm to 100 μm. And about a channel (1), length is 1 mm-100 mm, for example, Preferably it is 5 mm-70 mm, More preferably, it is 10 mm-50 mm. The channel (2) is, for example, 1 mm to 100 mm, preferably 5 mm to 70 mm, and more preferably 10 mm to 50 mm. About channel (3), they are 1 mm-500 mm, for example, Preferably they are 5 mm-200 mm, More preferably, they are 10 mm-100 mm. The channel (4) is, for example, 1 mm to 1000 mm, preferably 5 mm to 500 mm, and more preferably 10 mm to 200 mm.

  Channels (1) and (2) are preferably connected at the end of channel (3).

  The sample introduction microchannel (4) may be connected to or separated from the separation microchannel (3) at an arbitrary position. The sample introduction microchannel (4) may include a microstructure or a microspace necessary for sample preparation or pretreatment, or may be connected to another microchannel. For example, a minute space for filling fine particles necessary for sample purification, a minute space as a reaction vessel for performing sample amplification, a mixing vessel for mixing and reacting with various reaction solutions, and for mixing These include a microchannel and a structure for restricting the movement of the reaction solution.

  Among such channels (1) to (4), at least the separation microchannel (3) is filled with a buffer containing a polymer material (polymer matrix) having a molecular sieving ability. Buffers can be obtained by methods known in the art, for example, mechanical injection using pumping, vacuum pumping, or using electrokinetic phenomena or physical phenomena such as gravity, centrifugal force, etc. Can be introduced. A syringe pump, a vacuum pump, a diaphragm pump, or the like can be used as the pump. The pump can also be built into a microfluidic system using commonly known MEMS (microelectromechanical system) technology. As the buffer, well-known buffers such as Tris-acetate buffer and Tris-borate buffer can be used. In particular, in the separation method of the present invention, separation is expected to be performed at 30 ° C. or higher. Therefore, it is preferable to select a buffer solution having a buffer capacity even at a high temperature of 30 ° C. or higher. As the polymer matrix having molecular sieving ability, a known polymer matrix such as cellulose derivatives such as hydroxyethyl cellulose and hydroxymethyl cellulose, polyethylene oxide, polyacrylamide, polyvinyl pyrrolidone and the like can be used. Moreover, for example, a polymer matrix as disclosed in International Publication No. 2005/049196 can be used. The concentration of the polymer matrix in the buffer solution is, for example, 0.1% to 10% by mass, although it depends on the type and average molecular weight of the polymer matrix and the type of sample. For example, when a 200 bp DNA sample is used and hydroxyethyl cellulose having an average molecular weight of around 100,000 is used as the polymer matrix, a concentration of 0.1 to 3% by mass is suitable.

  In the microfluidic device used in the method of the present invention, the step (c) usually uses electrophoresis. In the steps (a) to (d), pumping by pump or electroosmotic flow can be used. Below, the case where it delivers using an electroosmotic flow is demonstrated to an example. Therefore, electrodes and a power source necessary for electrophoresis, generation of electroosmotic flow, and the like are appropriately provided.

  Further, in the microfluidic device used in the method of the present invention, at the end of each microchannel, a device for feeding liquid (pump, etc.), a filling unit for liquid and sample, etc. are appropriately provided as necessary. It is what has been.

  Next, one of the channels (1) and (2) is filled with a first liquid containing a denaturing agent and the other with no denaturing agent or at a concentration lower than that of the first liquid denaturing agent. The second liquid containing is basically introduced simultaneously to form a denaturant concentration gradient in the separation microchannel (step (a)).

  As the denaturing agent, a denaturing agent known in the art, for example, urea, formamide and formaldehyde, guanidine hydrochloride, acidic substance, basic substance and the like, or a mixture thereof can be used as appropriate. And such a denaturant can be used for the liquid containing a denaturant, for example using the said buffer solution.

  If the density | concentration of the modifier | denaturant of a 1st liquid and a 2nd liquid differs, there will be no restriction | limiting in particular. The concentration of the denaturant in the first liquid and the second liquid can be appropriately selected based on information such as the type of sample and preliminary examination. Moreover, one liquid may not contain a modifier.

  In order to form a concentration gradient of the denaturant, it is necessary to change the introduction amounts of the first liquid and the second liquid over time. Thereby, a change with time of the denaturant concentration is formed by mixing the first liquid and the second liquid at the connection points of the channels (1) to (3).

Method Form 1.
In one embodiment of the method of the present invention, the change over time of the introduction amounts of the first liquid and the second liquid in the step (a) is caused by the electrical properties of the liquid introduction microchannels (1) and (2). This is performed by changing the voltage for generating the osmotic flow with time, changing the flow rate of the pump with time, or changing both the voltage and the flow rate of the pump with time (step (a)). ). Thereby, a denaturant concentration gradient is formed in the separation microchannel.

In this embodiment, during the step (a), the temperature of the portion where the liquid introduction microchannels (1) and (2) are located is 30 to 80 ° C. Preferably, during the step (a), the temperature of the portion where the microchannels (1) and (2) are located is 40 to 70 ° C. or 50 to 60 ° C.
By setting it as such temperature, the introduction speed | rate of a 1st liquid and a 2nd liquid can be raised. Therefore, the time required for forming the concentration gradient of the denaturant is shortened, and the double-stranded nucleic acid can be separated efficiently. After the step (a), the temperature of the portion where the liquid introduction microchannels (1) and (2) are located is 10 to 30 ° C. Preferably, the temperature is 10 to 30 ° C. during the steps (b) to (c). Thereby, highly sensitive detection with good reproducibility in the process of (d) becomes possible.

  Next, double-stranded nucleic acid is introduced from the sample introduction microchannel into the separation microchannel (step (b)).

  There is no restriction | limiting in particular as a double stranded nucleic acid of a sample, A various nucleic acid can be used. Double-stranded nucleic acid electrophoresis in the presence of a denaturant concentration gradient can analyze a mixture of a plurality of nucleic acid samples, and can also analyze samples having the same chain length and different nucleic acid sequences. For example, biological samples such as human blood and cells, environmental samples such as food, soil, river water, and seawater, or nucleic acids extracted from activated sludge and methane fermentation sludge. The nucleic acid includes RNA and DNA, but preferably the nucleic acid is deoxyribonucleic acid (DNA), and more preferably, a predetermined region of DNA is artificially highly resistant to DNA denaturation at the end by polymerase chain reaction or the like. Double-stranded DNA amplified with a DNA sequence (GC clamp) is used.

  When the double-stranded nucleic acid of the sample is a solution, it may be used as a sample as it is. In addition, when the double-stranded nucleic acid of the sample is not a solution or when the solvent may adversely affect the analysis, a sample dissolved in an aqueous solvent can be used as the sample. The solvent can be selected from water and a buffer solution. Preferably, the double-stranded nucleic acid is dissolved in a buffer solution having a pH of 6-9. The buffer solution can be selected from a trishydroxyethylaminomethane-hydrochloric acid buffer solution or a good buffer conventionally used in the field of genetic engineering. As an additive, ethylenediaminetetraacetic acid or the like can be added to the sample. Since the concentration of the double-stranded nucleic acid depends on the detection method and the detection sensitivity of the detection apparatus, it can be determined by specifications or preliminary examination.

  In the step (b), it is preferable that no higher-order structural change occurs due to denaturation of the double-stranded nucleic acid. When the sample to be introduced is exposed to denaturing conditions specified by temperature and denaturant concentration, separation of double-stranded nucleic acids begins in the microchannel for sample introduction, preventing proper introduction of uniform samples. It is done. In addition, under the condition that the nucleic acid is denatured to a single-stranded nucleic acid, different types of nucleic acids form double strands (a heteroduplex is formed), which may reduce the reproducibility of the separation results. Therefore, during the step (b), it is preferable that the temperature of the portion where the sample introduction microchannel is located is equal to or lower than the denaturation temperature of the double-stranded nucleic acid to be analyzed. For example, when the denaturant concentration is 80% in a 200 bp DNA sample, the temperature is preferably 80 ° C. or lower, 60 ° C. or lower, or 40 ° C. or lower. For example, the temperature is 10 to 80 ° C., 10 to 60 ° C., 20 to 40 ° C., or 10 to 30 ° C.

  The introduced nucleic acid sample is electrophoresed in the denaturant concentration gradient in the separation microchannel, moves in a predetermined direction, and is separated (step (c)).

  The direction of sample movement in the separation microchannel where a denaturant concentration gradient exists is the type, molecular weight, and arrangement of double-stranded nucleic acids used in the analysis, the material of the microfluidic device, the magnitude of the electric field strength, and the pressure gradient. It depends on etc. Therefore, the part where the double-stranded nucleic acid is separated and the part where the separated double-stranded nucleic acid is detected can also be changed.

  FIG. 1 shows a form in which the double-stranded nucleic acid is moved and separated from the position where the double-stranded nucleic acid is introduced in the direction opposite to the direction in which the liquid-introducing microchannel exists.

  FIG. 2 shows a form in which the double-stranded nucleic acid is moved and separated from the position where the double-stranded nucleic acid is introduced in the direction in which the liquid introduction microchannel exists.

  During the step (c), the temperature of the portion where the separation microchannel is located and the portion where the introduced double-stranded nucleic acid is separated is preferably 30 to 70 ° C. By setting such a temperature, the separation performance is improved. It is known that urea is thermally decomposed at 70 ° C. or higher.

  Next, the separated double-stranded nucleic acid is detected (step (d)).

  The detection is selected from well-known methods such as fluorescence detection, luminescence detection, absorption detection, and electrochemical detection, which are commonly used nucleic acid detection means, by appropriate examination from the application, cost, sensitivity, accuracy, and device shape. The When performing fluorescence detection, for example, a method of fluorescently labeling a double-stranded nucleic acid with an oligonucleotide used for PCR reaction, a method of labeling with a fluorescent labeling substance in advance, or electrophoresis of a nucleic acid stain is performed. There are ways to add it to the solution. As the detector, a camera, a photomultiplier tube, a UV detector, a photodiode detector, or the like can be used.

  During the step (d), the temperature of the portion where the separation microchannel is located and where the separated double-stranded nucleic acid exists, that is, the detection portion is kept lower than the temperature of the step (c). It is preferable. Preferably, it is constant in a temperature range of 10 to 50 ° C. or a temperature range of 10 to 30 ° C. By setting such a temperature, the detection sensitivity is improved.

  In this embodiment in which the temperature of the portion where the liquid introduction microchannels (1) and (2) are located is changed over time, the double-stranded nucleic acid shown in FIG. 2 moves in the direction in which the liquid introduction microchannel exists. Particularly suitable in the case of separated forms. In the case of the form shown in FIG. 2, the double-stranded nucleic acid moves and is separated in the direction in which the liquid introduction microchannel is present, so that the detection portion is close to the position where the liquid introduction microchannel is present. . Therefore, by setting the temperature in the vicinity of the detection portion, that is, the portion where the liquid introduction microchannel is located, to 10 to 30 ° C., it is possible to prevent the detection sensitivity from being lowered or to improve the detection sensitivity. .

Method form 2.
In WO 2005/049196, the change over time of the introduction amount of the first liquid and the second liquid changes the voltage for the generation of electroosmotic flow in the channels (1) and (2) over time. It is disclosed that this can be done.

  In another embodiment of the method of the present invention, the change over time of the introduction amounts of the first liquid and the second liquid in the step (a) is caused in the portion where the channels (1) and (2) are located. This can be done by changing the temperature over time. That is, the temperature of the portion where one of the liquid introduction microchannels (for example, the channel (1)) is located is set to a relatively high temperature (for example, 80 ° C.), and the other microchannel (for example, the channel (2) )) Is set to a relatively low temperature (for example, 20 ° C.). Then, with the introduction of the first liquid and the second liquid, over time, the high temperature is changed to a low temperature (eg, 80 ° C. to 20 ° C.), and at the same time, the low temperature is changed to a high temperature (eg, 20 ° C. to 80 ° C.). By changing. Since the viscosity of the liquid is temperature-dependent, when the voltage for generating the electroosmotic flow is constant, the moving speed of the liquid increases, that is, the introduction amount increases as the viscosity of the liquid decreases. Therefore, as described above, the viscosity of the liquid can be changed over time by changing the temperature of the portion where the microchannel is located over time. The rate of change in temperature at this time can be determined by preliminarily examining the type of nucleic acid sample, the type of polymer matrix, the analysis time, and the like. As a result, the introduction amounts of the first liquid and the second liquid can be changed over time, so that a concentration gradient of the denaturant can be formed. In general, the higher the temperature, the lower the viscosity. Therefore, by setting the temperature of the portion where the liquid-introducing microchannel is located to 30 ° C. to 80 ° C., a value higher than room temperature, the first The introduction speed of the liquid and the second liquid can be increased from the introduction conditions at room temperature. Therefore, the time required for forming the concentration gradient of the denaturant is reduced, the time required for separating the double-stranded nucleic acid as a whole is reduced, and the analysis can be performed in a short time.

  The amount of change in the temperature of the liquid-introducing microchannel is preferably 20 ° C. to 90 ° C., and more preferably 30 ° C. to 80 ° C., which is practically effective.

  In one embodiment of the present invention, during the step (a), the temperature of the portion where one of the microchannels for introducing liquid is located is raised from 20 ° C. to 90 ° C., and at the same time the other microchannel is located. The temperature of the portion to be reduced is lowered from 90 ° C. to 20 ° C. Alternatively, during the step (a), the temperature of the portion where one microchannel of the liquid introduction microchannel is located is increased from 30 ° C. to 80 ° C., and at the same time, the temperature of the portion where the other microchannel is located is 80 ° C. To 30 ° C. Alternatively, during the step (a), the temperature of the portion where one of the microchannels for introducing liquid is located is increased from 30 ° C. to 60 ° C., and the temperature of the portion where the other microchannel is located is simultaneously raised to 60 ° C. To 30 ° C.

  In addition, the temperature of the portion located at the liquid introduction microchannel rises so that the viscosity of the liquid introduced into one microchannel becomes one third to one half, and at the same time, the other microchannel. It is preferable to change the viscosity of the liquid introduced into the liquid so as to decrease so that the viscosity becomes 2 to 3 times.

  The temperature change of each channel described above can be performed with a constant voltage of each channel for liquid feeding. It is also possible to change the temperature while changing the voltage of each channel in such a manner that the change over time in the amount of liquid introduced has the same tendency.

  The steps (b) to (d) following the step (a) are performed in the form 1. Can be carried out as described in.

  In the method of the present invention, as described above, it is necessary to control the temperature of the portion where each microchannel is located. Such temperature control can be performed using, for example, a temperature control device including a heating / cooling device and a temperature detection device connected to a control device including a CPU having an arithmetic function. That is, along the microchannel where temperature control is required, an appropriate heating / cooling device, cooling device, and temperature detection device are provided on the upper surface and / or lower surface of the microfluidic device body, and the temperature of that portion is controlled by the control device. Can be done.

  In the method of the present invention, the “positioned portion” in the description of the portion where the liquid introduction microchannel is located, etc., is a region including at least a portion of the channel where temperature control is intended and the vicinity thereof. Means an area that does not include other channels. The size of such a “positioned portion” can be appropriately changed by selecting the size of the heating / cooling device used for temperature control.

Accordingly, the present invention relates to a microfluidic system having temperature control means for controlling the temperature of a portion where a microchannel is located.

  The temperature control means includes, for example, a control device including a CPU having a calculation function, and a heating / cooling device and a temperature detection device connected to the control device.

  As the heating / cooling device, for example, a general heater, a Peltier element, or the like can be used. If a Peltier element is used, heating and cooling can be performed, so that more responsive and precise temperature control over a wide range is possible. Alternatively, a metal thin film having high electrical resistance may be directly patterned on the microfluidic device main body by plating, vapor deposition, sputtering, ion plating, sticking, etc., and provided as a heater. A transparent conductive film such as indium tin oxide may be provided as a heater. The cooling means may use a fan, a refrigerator, or a heat exchanger, for example. By having an external cooling means, a higher range of temperature control is possible. Examples of the temperature detection device include a thermocouple, a resistance temperature detector, a thermistor, and the like.

  A radiation thermometer can also be used if it is difficult to fix the temperature detection device depending on the dimensions and material of the microfluidic device. Alternatively, a temperature detecting device may be provided by patterning a metal thin film having temperature dependence directly on the microfluidic device body by plating, vapor deposition, sputtering, ion plating, sticking, or the like.

  In the microfluidic system of the present invention, the heating / cooling device and the temperature detection device can be appropriately installed in a portion requiring temperature control. One or more heating / cooling devices and temperature detection devices may be installed in the microfluidic system. The size of the heating / cooling device can be set as appropriate. For example, the heating / cooling device can be a size that includes the entire portion where the separation microchannel is located, and the heating / cooling device can be a size that includes a portion of the portion where the separation microchannel is located. It can also be a device.

  FIGS. 3 and 4 and FIGS. 5 and 6 show an example of a microfluidic system using a Peltier element as a temperature detection device and a heating / cooling device. The device is connected to the control device). 3 and 5 are top views, and FIGS. 4 and 6 are cross-sectional views. The temperature detection device and the Peltier element are respectively installed on the upper surface and the lower surface of the microfluidic device. A temperature detecting device and a Peltier device are provided in the liquid introducing microchannel, the portion including the sample introducing microchannel, and the portion including the separation microchannel.

  In the configuration of FIG. 5, a pair of temperature detection devices and Peltier elements are installed in a portion including each liquid introduction microchannel and sample introduction microchannel. The separation microchannel is provided with two pairs of temperature detecting devices and an additional Peltier element. The temperature of the part is controlled by each pair of temperature detection devices and Peltier elements. In the configuration of FIG. 3, a pair of temperature detection devices and Peltier elements are installed for two liquid introduction microchannels.

  In FIG. 5, one temperature detection device and one Peltier element are provided in each part, but a plurality of Peltier elements and a plurality of Peltier elements may be provided in each part.

  The microfluidic system of FIG. 5 corresponds to the form of FIG. 1, that is, the form in which the microfluidic system is moved and separated from the position where the double-stranded nucleic acid is introduced in the direction opposite to the direction in which the liquid-introducing microchannel exists. is there. The microfluidic system of FIG. 3 corresponds to the configuration of FIG. 2, that is, the configuration in which the microfluidic system is moved and separated from the position where the double-stranded nucleic acid is introduced in the direction in which the liquid-introducing microchannel exists.

  In the microfluidic system of FIGS. 3 and 5, a temperature detecting device and a Peltier element are installed in two parts of the separation microchannel, and the parts where the double-stranded nucleic acids are separated (separation parts), respectively. In addition, the temperature of the detection portion can be controlled.

  In the microfluidic system of FIG. 5, temperature control means (a pair of temperature detection devices and Peltier elements) are installed in each liquid introduction microchannel, so that the temperature of the portion where each liquid introduction microchannel is located is independent. Can be controlled. Therefore, the system of FIG. And method form 2. It can be performed.

  In the microfluidic system of FIG. 3, since one temperature control means (a pair of temperature detecting devices and a Peltier element) is installed in two liquid introduction microchannels, the temperature of the portion where each liquid introduction microchannel is located It is controlled as a unit. Therefore, the above-described method mode 2. of the present invention is performed by the system of FIG. It can be performed.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

  The microfluidic device used has the shape shown in FIG. 2 and is made of acrylic resin. The length of the microchannels (1) and (2) was 21.5 mm, and the length of the microchannel (3) was 40 mm. The microchannel of (1) is filled with a tris-acetate buffer solution containing rhodamine B as a fluorescent substance and 1.75% of hydroxyethylcellulose as a polymer matrix, and 1.75% of hydroxyethylcellulose is loaded into the microchannel of (2). Filled with Tris-acetate buffer. The temperature of the entire microfluidic device was kept constant at 30 ° C. Electrodes are connected to the ends of the channels (1) to (4), and a high voltage power source is connected to each electrode. A potential of 3 KV was applied to the electrodes connected to the channels (1) and (2), and the electrode connected to the microchannel (3) was connected to the ground. Under this condition, the fluorescence of rhodamine B flowing into the microchannel (3) from the microchannel (1) was photographed with a CCD camera for microscope. When the moving speed of rhodamine B was calculated from the obtained moving image, it was about 20 μm / sec.

  Accordingly, when the actual denaturant concentration gradient is 50 mm, the time required for forming the denaturant concentration gradient is 42 minutes.

  When the temperature of the microchannel of (1) and (2) is 60 ° C., the viscosity of hydroxyethyl cellulose is about one-third that of 30 ° C., and the electroosmotic flow rate is also about one-third. It becomes. Therefore, even when the same voltage is applied, the time required for forming the denaturant concentration gradient is 14 minutes. If the time required for introduction of the nucleic acid sample is 1 minute and the separation time by electrophoresis is 15 minutes, the total analysis time is about half from 58 minutes to 30 minutes.

FIG. 1 is a schematic view showing a microfluidic device. FIG. 2 is a schematic view showing a microfluidic device. FIG. 3 is a top view of a microfluidic system provided with temperature control means. FIG. 4 is a cross-sectional view of a microfluidic system provided with temperature control means. FIG. 5 is a top view of a microfluidic system provided with temperature control means. FIG. 6 is a cross-sectional view of a microfluidic system provided with temperature control means.

Explanation of symbols

  (1) is a microchannel for liquid introduction.

  (2) is a microchannel for liquid introduction.

  (3) is a separation microchannel.

  (4) is a microchannel for sample introduction.

Claims (10)

Microchannel for separation,
Two liquid introduction microchannels connected to the separation microchannel, and a sample introduction microchannel connected to the separation microchannel;
, By performing electrophoresis in the presence of a denaturant concentration gradient using a microfluidic device having at least a double-stranded nucleic acid based on a difference in base sequence,
The method
at least,
(A) A first liquid containing a denaturant is introduced into one microchannel of the two liquid introduction microchannels, and at the same time, the concentration is lower than the concentration of the denaturant of the first liquid into the other microchannel. Introducing a second liquid containing or not containing a denaturant to form a denaturant concentration gradient in the separation microchannel,
(B) introducing a double-stranded nucleic acid from the sample introduction microchannel into the separation microchannel;
(C) separating the introduced double-stranded nucleic acid, and (d) detecting the separated double-stranded nucleic acid,
Including
The method according to claim 1, wherein the temperature of the microchannel for liquid introduction is controlled during the step (a). Microchannel for separation,
Two liquid introduction microchannels connected to the separation microchannel, and a sample introduction microchannel connected to the separation microchannel;
, By performing electrophoresis in the presence of a denaturant concentration gradient using a microfluidic device having at least a double-stranded nucleic acid based on a difference in base sequence,
The method
at least,
(A) A first liquid containing a denaturant is introduced into one microchannel of the two liquid introduction microchannels, and at the same time, the concentration is lower than the concentration of the denaturant of the first liquid into the other microchannel. Introducing a second liquid containing a denaturant or not containing a denaturant to form a denaturant concentration gradient in the separation microchannel,
(B) introducing a double-stranded nucleic acid from the sample introduction microchannel into the separation microchannel;
(C) separating the introduced double-stranded nucleic acid, and (d) detecting the separated double-stranded nucleic acid,
Including
The method, wherein a temperature of a portion where the liquid introduction microchannel is located is set to 30 to 80 ° C. during the step (a). Microchannel for separation,
Two liquid introduction microchannels connected to the separation microchannel, and a sample introduction microchannel connected to the separation microchannel;
, By performing electrophoresis in the presence of a denaturant concentration gradient using a microfluidic device having at least a double-stranded nucleic acid based on a difference in base sequence,
The method
at least,
(A) A first liquid containing a denaturant is introduced into one microchannel of the two liquid introduction microchannels, and at the same time, the concentration is lower than the concentration of the denaturant of the first liquid into the other microchannel. Introducing a second liquid containing a denaturant or not containing a denaturant to form a denaturant concentration gradient in the separation microchannel,
(B) introducing a double-stranded nucleic acid from the sample introduction microchannel into the separation microchannel;
(C) separating the introduced double-stranded nucleic acid, and (d) detecting the separated double-stranded nucleic acid,
Including
During the step (a), the temperature of the portion where one of the microchannels for liquid introduction is located is raised, and the temperature of the portion where the other microchannel is located is lowered at the same time, Said method.   The method according to claim 2, wherein after the step (a), the temperature of the portion where the liquid introduction microchannel is located is set to 10 to 30 ° C.   5. The method according to claim 2, wherein a temperature of a portion where the liquid introduction microchannel is located is set to 10 to 30 ° C. during the steps (b) to (c).   4. The method according to claim 2, wherein a temperature of a portion where the sample introduction microchannel is located is set to 10 to 30 ° C. during the step (b). 5.   During the step (d), the temperature of at least a part of the part where the separation microchannel is located and the part where the separated double-stranded nucleic acid is present is 10 to 50 ° C. The method according to claim 2 or claim 3.   During the step (a), the temperature of the portion where one microchannel of the liquid introduction microchannel is located is increased, and the temperature of the portion where the other microchannel is located is lowered at the same time. The method according to claim 3, wherein the temperature change of the microchannel is 30 ° C. to 80 ° C.   During the step (a), the temperature of the portion where one of the liquid introduction microchannels is located is adjusted so that the viscosity of the liquid introduced into the one microchannel is one third to one half. And at the same time, the temperature of the portion where the other microchannel is located is lowered so that the viscosity of the liquid introduced into the other microchannel is two to three times, The method of claim 3. Microchannel for separation,
Two liquid introduction microchannels connected to the separation microchannel,
A sample introduction microchannel connected to the separation microchannel,
At least one temperature control means for controlling the temperature of at least a part of the portion where the separation microchannel is located;
At least one temperature control means for controlling the temperature of at least a part of the portion where the two liquid introduction microchannels are located, and controls the temperature of at least a part of the portion where the sample introduction microchannel is located At least one temperature control means,
And a microfluidic system for separating double-stranded nucleic acids based on the difference in base sequence by performing electrophoresis in the presence of a denaturant concentration gradient.

Images