JP2003222611A - Separating apparatus and method therefor, and manufacturing method thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a separating technique that can quickly separate a sample with improved resolution by a small amount of sample and has less problems of blocking or the like. <P>SOLUTION: Many hydrophobic regions 705 are arranged at a channel where a sample passes at an nearly equal interval, and a region other than the hydrophobic regions 705 is in structure where the surface of a hydrophilic substrate 701 is exposed. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an apparatus and a separation method for separating samples having different sizes, polarities, affinity for water and the like.

[0002]

2. Description of the Related Art In the analysis of nucleic acids and proteins, the operations of separating and purifying a sample in advance or separating and analyzing the sample according to size and charge are frequently performed. For example, in the Maxam-Gilbert method, which is widely used as a nucleotide sequence determination method, one end of DNA is labeled with ³² P, this is chemically decomposed to obtain fragments of various lengths, and then electrophoresed. After that, the process of separating and then performing autoradiography to read the base sequence is performed. Such separation operation is an important factor that determines the length of analysis time, and shortening the time required for separation is an important technical issue in this field. In order to solve such a technical problem, development of a separation device capable of accurately separating a desired substance in a short time is desired.

As such a separation device, a capillary electrophoresis device has heretofore been widely used. However, capillary electrophoresis requires a long time for measurement and requires a large amount of sample. Also, the resolution is not always at a satisfactory level.

On the other hand, as a device for separating a target substance, US Pat. No. 5,837,115 (Patent Document 1) discloses a separating device in which a large number of obstacles are arranged in an array. Examples of the separation target include cells, viruses, macromolecules, and microparticles.

[0005]

[Patent Document 1] US Pat. No. 5,837,115

[0006]

However, this technique still has room for improvement in the following points. Primarily,
Since there are cases where clogging occurs due to macromolecules and particles, there is a limit to improvement in throughput. Secondly, there is a problem that processing cost is required to process many obstacles.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a separation apparatus and a separation method capable of accurately separating a desired substance with a small amount of sample in a short time.

[0008]

According to the present invention, there is provided a separation device including a substrate, a flow channel formed on the surface of the substrate and through which a sample passes, and a sample introduction section and a sample separation section provided in the flow channel. An apparatus is provided, wherein the surface of the sample separation unit has a hydrophilic region and a hydrophobic region.

According to the present invention, the substrate, the flow channel formed on the surface of the substrate through which the sample passes, the sample introducing section and the sample discharging section provided in the flow channel, and the sample introducing section to the sample discharging section. A separation device comprising a sample separation section provided in a flow path up to the first separation area, wherein the surface of the sample separation section has a plurality of first areas arranged apart from each other and the first area. And a second region occupying the surface of the sample separation portion except for, one of the first region and the second region is a hydrophobic region, the other is a hydrophilic region A separation device is provided.

In this separating device, the first regions may be two-dimensionally arranged at substantially equal intervals.
That is, the first regions can be regularly arranged in the vertical and horizontal directions at substantially equal intervals. The separating apparatus may further include an external force applying means so that the sample is moved from the sample introducing section to the sample discharging section by an external force. The type of external force is an electric field,
Surface tension, pressure and the like can be used, and examples of the external force applying means include a voltage applying section and a pump. When surface tension is selected as the type of external force, no special external force applying means may be provided.

According to the separation device of the present invention, (i) the first region is a hydrophobic region and the second region is a hydrophilic region. (Ii) the first region is a hydrophilic region, It is possible to employ either of the configurations in which the second region is a hydrophobic region. The hydrophilic region in the present invention means that it is more hydrophilic than the hydrophobic region. The degree of hydrophilicity can be grasped, for example, by measuring the water contact angle.

The principle of the separation apparatus according to the present invention will be described below by taking the case of (i) above as an example. in this case,
The sample to be separated is introduced into the apparatus in a state of being dissolved or dispersed in a solvent having relatively high hydrophilicity. Such a solvent is distributed only in the hydrophilic region (second region) while avoiding the surface of the hydrophobic region (first region) in the sample separation part. Therefore, the gap between the hydrophobic regions becomes a path through which the sample to be separated passes, and as a result, the time required for passage through the sample separation unit is determined by the relationship between the interval between the hydrophobic regions and the sample size. Becomes Thereby, the sample is separated according to the size.

On the other hand, in the present invention, in addition to separation according to size, separation according to the polarity of the sample is also performed. That is, it is possible to separate a plurality of types of samples having different degrees of hydrophilicity / hydrophobicity. In the example of (i) above, a highly hydrophobic sample is easily captured in the hydrophobic region and the outflow time is relatively long, while a highly hydrophilic sample is difficult to be captured in the hydrophobic region and the outflow time is relatively short. Become. As described above, according to the present invention, not only the size of the sample but also the polarity is separated, and it is possible to realize the separation of a multi-component system which has been difficult to separate in the related art.

The separating apparatus according to the present invention uses a sample separating section provided on the surface of the flow path as a separating means, unlike a method of separating by a structure which becomes an obstacle. In the case of membrane separation, it is necessary to precisely control the size of pores in the membrane, but it is not always easy to stably produce a membrane having pores of a desired size and shape. On the other hand, the present invention can form the sample separation portion by the surface treatment of the flow channel, and the desired separation performance can be obtained by controlling the interval of the first region. The device configuration can be realized relatively easily. For example,
The sample separation part of the device of the present invention can be prepared by depositing a compound having a hydrophobic group in the mask opening, and in this case, the interval between the hydrophobic regions can be easily adjusted by adjusting the mask opening width. it can. That is, the interval between the hydrophobic regions can be appropriately adjusted according to the purpose of separation, and the sample separation unit can be configured according to the purpose of separation. In particular, in the separation of proteins and DNAs, it is required to separate substances of various sizes from separation of substances of huge size to separation of substances of nano order. Among them, it has been extremely difficult to separate a nano-order substance with high resolution in a short time by the conventional technique. In the separation device according to the present invention, the separation size can be narrowed by narrowing the interval between the first regions. The space between the first regions can be easily realized by using a microfabrication technique, so that it is possible to preferably realize the separation of the nano-order size substance.

Further, according to the separation apparatus of the present invention, it is possible to perform separation in a short time with a small amount of sample. The separation according to the present invention is performed by the surface characteristics of the sample separation unit, so that it is possible to realize precise separation, and since the loss of the sample is small, it is possible to realize a sufficiently high resolution even with a small amount of sample, and Excellent resolution can be realized. Furthermore, since the separation apparatus according to the present invention performs separation according to the surface characteristics of the flow path that passes through the sample, there are few problems such as clogging. Further, after use, the surface of the sample separation part can be cleaned very easily by a method such as flowing a cleaning liquid.

The separation device of the present invention can realize separation of various functions depending on the relationship between the distance between the adjacent first regions and the sample size of the separation target contained in the liquid. When the sample size is larger than the above distance, the separating device functions as a sample concentrating device. The sample separation unit functions as a filter, and the sample is dammed upstream of the sample separation unit. As a result, the sample is concentrated to a high concentration on the upstream side of the sample separation unit.

On the other hand, when the sample size is smaller than the above distance, the sample separating section performs a sample separating function, and the sample is separated in the sample separating section according to the size and the degree of hydrophilicity. As a result, the separated sample will flow out to the downstream side of the sample separation unit.

Further, according to the present invention, there is provided a sample separation method characterized in that the above-mentioned separation device is used to introduce a sample from the sample introduction part to separate a predetermined component in the sample.

According to this sample separation method, highly accurate sample separation can be realized while solving problems such as clogging.

Further, according to the present invention, there is provided a method of manufacturing a separation device comprising a substrate, a flow path for a sample formed on the surface of the substrate, and a sample separation section provided in the flow path, After the step of forming a channel having a hydrophilic surface, and providing a mask having an opening on at least a part of the surface of the channel,
A step of depositing a compound having a hydrophobic group on the surface of the flow channel from the opening and then removing the mask to form a sample separation part in which a hydrophobic region is arranged. A method of manufacturing a device is provided. Here, the flow channel can also be provided by forming a groove in a substrate having a hydrophilic surface. In addition, in the step of forming the sample separation portion, a hydrophobic region that becomes the side wall of the flow channel can be formed at the same time. Furthermore, the hydrophobic region of the sample separation section may be configured such that a plurality of hydrophobic regions are arranged separately. In this case, the mask can be formed to have a plurality of openings. According to the above-mentioned manufacturing method, a pattern in which a hydrophobic surface and a hydrophilic surface are mixed can be manufactured with high yield and high accuracy. In the method for manufacturing the separation device, the first regions may be two-dimensionally arranged at substantially equal intervals. That is, the first regions can be regularly arranged in the vertical and horizontal directions at substantially equal intervals.

Further, according to the present invention, there is provided a method of manufacturing a separation device comprising a substrate, a flow path for a sample formed on the surface of the substrate, and a sample separation section provided in the flow path, After forming a channel on a substrate having a hydrophobic surface and providing a mask having an opening on at least a part of the surface of the channel, a compound having a hydrophilic group is deposited on the surface of the channel from the opening. And then removing the mask to form a sample separation portion in which the hydrophilic region is arranged, and a method for manufacturing a separation device is provided. Here, the flow path can also be provided by forming a groove in a substrate having a hydrophobic surface. Further, the hydrophilic region of the sample separation part may be configured such that a plurality of hydrophilic regions are arranged separately. In this case, the mask can be formed to have a plurality of openings. According to the above-mentioned manufacturing method, a pattern in which a hydrophobic surface and a hydrophilic surface are mixed can be manufactured with high yield and high accuracy. In the method for manufacturing the separation device, the first regions may be two-dimensionally arranged at substantially equal intervals. That is, the first regions can be regularly arranged in the vertical and horizontal directions at substantially equal intervals. For the compound having a hydrophobic group and the compound having a hydrophilic group, for example, a silane coupling agent can be used.

According to the present invention, there is provided a method of manufacturing a separation device comprising a substrate, a sample passage formed on the surface of the substrate, and a sample separation section provided in the passage, the method comprising: Separation, comprising: forming a flow channel having a porous surface; and forming a sample separation section in which a liquid silicone compound is attached to the surface of the flow channel to arrange a hydrophobic region. A method of manufacturing a device is provided. Here, the flow channel can also be provided by forming a groove in a substrate having a hydrophilic surface. In addition, in the step of forming the sample separation portion, a hydrophobic region that becomes the side wall of the flow channel can be formed at the same time. Furthermore, the hydrophobic region of the sample separation section may be configured such that a plurality of hydrophobic regions are arranged separately. In this case, the mask can be formed to have a plurality of openings. According to the above manufacturing method, a pattern in which a hydrophobic surface and a hydrophilic surface are mixed can be easily manufactured with high accuracy.

In this manufacturing method of the present invention, the step of forming the sample separation part may include a step of bringing the surface of the silicone resin containing the liquid silicone compound into contact with the channel surface. Here, as the liquid silicone compound, for example, silicone oil can be used. According to this method, a pattern in which a hydrophobic surface and a hydrophilic surface are mixed can be formed by a simple process.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the sample separation section is
It can be prepared by a method such that a part of the substrate having a hydrophilic surface is subjected to a hydrophobic treatment or a part of the substrate having a hydrophobic surface is subjected to a hydrophilic treatment, and the substrate surface is subjected to both a hydrophobic treatment and a hydrophilic treatment. It can also be made.

As the substrate having a hydrophilic surface, a quartz substrate or a glass substrate can be used. As the substrate having a hydrophobic surface, a resin substrate such as silicone resin or polyethylene resin can be used.

In the hydrophobic treatment or hydrophilic treatment, a compound having a structure having both a unit that adsorbs or chemically bonds with a substrate material and a unit that has a hydrophobic or hydrophilic decorative group in a molecule is attached or bonded to the substrate surface. It is realized by As such a compound, for example, a silane coupling agent or the like can be used.

As the silane coupling agent, vinyltrichlorosilane, vinyltrimethoxysilane, vinyltriethoxysilane, β- (3,4 epoxycyclohexyl)
Ethyltrimethoxysilane, γ-glycidoxypropyltrimethoxysilane, γ-glycidoxypropylmethyldiethoxysilane, γ-glycidoxypropyltriethoxysilane, γ-methacryloxypropylmethyldimethoxysilane, γ-methacryloxypropyl Trimethoxysilane, γ-methacryloxypropylmethyldiethoxysilane, γ-methacryloxypropyltriethoxysilane, N-β (aminoethyl) γ-aminopropylmethyldimethoxysilane, N-β (aminoethyl) γ-aminopropyltri Methoxysilane, N-β (aminoethyl)
γ-aminopropyltriethoxysilane, γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, N-phenyl-γ-aminopropyltrimethoxysilane, γ-chloropropyltrimethoxysilane, γ-mercaptopropyltrimethoxysilane Silane, 3-
Isocyanatopropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, 3-triethoxysilyl-N- (1,3-dimethyl-butylidene), 3-thiolpropyltriethoxysilane and the like can be mentioned.

Among the above, preferred as the silane coupling agent having a hydrophilic group are those having an amino group, and specifically, N-β (aminoethyl) γ-
Aminopropylmethyldimethoxysilane, N-β (aminoethyl) γ-aminopropyltrimethoxysilane, N
-Β (aminoethyl) γ-aminopropyltriethoxysilane, γ-aminopropyltrimethoxysilane, γ-
Aminopropyltriethoxysilane, N-phenyl-γ
-Aminopropyltrimethoxysilane and the like.

Preferred examples of the silane coupling agent having a hydrophobic group include those having a thiol group, and specific examples thereof include 3-thiolpropyltriethoxysilane.

As a method of applying the coupling agent liquid, etc.,
Spin coating method, spraying method, dipping method, vapor phase method and the like are used. Spin coating is a coupling agent, etc.
In this method, a liquid in which the constituent materials of the binding layer are dissolved or dispersed is applied by a spin coater. According to this method, the film thickness controllability becomes good. The spray method is a method of spraying a coupling agent solution or the like onto a substrate, and the dipping method is a method of immersing the substrate in the coupling agent solution or the like. According to these methods, a film can be formed by a simple process without requiring a special device. The vapor phase method is a method in which the substrate is heated as necessary, and vapor such as a coupling agent liquid is caused to flow there. Also by this method, a thin film can be formed with good film thickness controllability. Among these, the method of spin-coating the silane coupling agent solution is preferably used. This is because excellent adhesiveness can be stably obtained. At this time, the concentration of the silane coupling agent in the solution is preferably 0.01 to 5 v.
/ v%, more preferably 0.05 to 1 v / v%. As the solvent for the silane coupling agent solution, pure water; alcohols such as methanol, ethanol, isopropyl alcohol; esters such as ethyl acetate can be used alone or in admixture of two or more. Of these, ethanol, methanol, and ethyl acetate diluted with pure water are preferable. This is because the effect of improving the adhesiveness becomes particularly remarkable. After applying the coupling agent liquid or the like, it is dried. The drying temperature is not particularly limited, but is usually room temperature (25 ° C.) to 170 ° C. The drying time depends on the temperature, but is usually 0.5
~ 24 hours. The drying may be performed in air, but may be performed in an inert gas such as nitrogen. For example, a nitrogen blowing method in which nitrogen is blown onto the substrate to dry it can be used.

In the present invention, the shape of the first region is not particularly limited and includes various shapes such as a circle, an ellipse, a quadrangle and a triangle. Further, it may have a convex shape having a predetermined height by a hydrophobic surface treatment. The size of the first region is also not particularly limited and is appropriately selected according to the purpose and application of the separation device. INDUSTRIAL APPLICABILITY The separation device of the present invention can be suitably used for separation / purification of materials having different sizes and polarities in a fluid. It is particularly suitable for separating biological substances. For example, it is suitable for using blood or saliva of humans or other animals as a sample and separating and concentrating the following components. (i) Separation and concentration of cells and other components (ii) Separation and concentration of solids (fragment of cell membrane, mitochondria, endoplasmic reticulum) and liquid fraction (cytoplasm) among components obtained by disrupting cells ( iii) Of the components of the liquid fraction, high molecular weight components (DNA,
Separation of RNA, protein, sugar chain) and low molecular weight components (steroids, glucose, etc.), and concentration (iv) Separation of polymer degradation products and undegradation products The separation device according to the present invention also separates substances of minute size. It is possible and applicable to separation / purification of nucleic acids such as nucleic acid fragments of various sizes, or organic molecules such as amino acids, peptides and proteins, metal ions and the like. The interval between the first regions in the present invention is appropriately set according to the purpose of separation. For example, (i) separation of cells and other components, concentration (ii) separation of solids (fragments of cell membranes, mitochondria, endoplasmic reticulum) and liquid fractions (cytoplasm) of components obtained by disrupting cells, Concentration (iii) Of the components of the liquid fraction, high molecular weight components (DNA,
In the processing such as separation and concentration of RNA, protein, sugar chain) and low molecular weight components (steroid, glucose, etc.), in the case of (i), 1 μm to 10 μm
m, (ii), 100 nm to 1 μm, (iii)
1 nm to 100 nm.

The separation apparatus of the present invention can be used as an analysis apparatus by providing a detection section on the downstream side of the sample separation section. Also,
It is also possible to adopt a configuration in which a predetermined component is collected from the sample discharging section.

In the present invention, the distance between the first regions is appropriately set according to the purpose of separation. For example, it can be 100 nm or less. Since the hydrophobic region can be formed by a film forming process utilizing a lithography technique such as electron beam exposure, the hydrophobic region is 100 nm or less, and further 5
Those with a thickness of 0 nm or less can also be realized. As a result, it is possible to realize separation of components, which has been difficult in the past.

In the separation apparatus of the present invention, it is possible to adopt a configuration in which a plurality of sample separation parts are included and a path for the sample to pass between adjacent sample separation parts is provided. When such a configuration is adopted, the sample is separated according to a principle different from that of a normal molecular sieve. With the separation apparatus of the present invention, it is possible to perform separation based on both the size and hydrophilicity / hydrophobicity of the sample, the affinity for water, and the polarity, but the following description will focus on size separation. In ordinary molecular sieves, the larger the molecular size of a substance, the greater the degree of obstruction of passage by the sieve. Therefore, the large size substance is separated after being discharged later than the small size substance. On the other hand, in the device of the present invention, the smaller the sample size, the longer the path through the sample separation section. Therefore, the small size substance is discharged later than the large size substance. Is separated by. A large-sized substance passes through the separation area relatively smoothly. As a result, the throughput in the separation operation is significantly improved. In particular, in the separation of nucleic acids, proteins, etc., the radius of gyration of the molecule also extends over a very wide range, so that the separation efficiency tends to decrease due to the huge size of the substance. According to the present invention, since such a problem is solved, it can be suitably applied to the separation of nucleic acids, proteins and the like.

In the present invention, the width of the path between the sample separation parts can be made larger than the average interval between the hydrophobic regions in the sample separation part. By doing so, the large-sized substance smoothly passes through the path part in the sample separation part, and the small-sized substance passes through the sample separation part, and the sample is separated after the long path depending on the size. It will pass the department. As a result, the separation in which the large-sized substance is discharged later than the small-sized substance is more smoothly performed.

Here, the interval between the first regions in the sample separation section can be set to an arbitrary value for each sample separation section. Therefore, according to the present invention, two kinds of parameters, that is, the distance between the first regions in the sample separation part and the width of the path can be set arbitrarily, which allows the occurrence of clogging even in a sample having a wide size distribution. It is possible to perform separation with high resolution without lowering the throughput and throughput. For example, in order to separate small-sized molecules with high resolution, the interval between the first regions is narrowed to the order of several nanometers to tens of nanometers, while the width of the path in the sample separation section is increased. Molecules of large size can be smoothly moved to prevent clogging and decrease in separation efficiency.

The first regions forming each sample separation portion may be formed to have substantially the same size and at equal intervals. By doing so, the sensitivity of separation in the sample separation section can be increased. When the number of the first regions in the sample separation section is large, the resolution is improved.

The sample separation part may be composed of first regions of different sizes. That is, it is also possible to adopt a configuration in which the first regions are formed with different sizes and intervals in each sample separation part. In this way, even for samples with a very wide size distribution,
Separation can be performed with high resolution without causing clogging or lowering of throughput.

In the separation apparatus of the present invention, it is possible to adopt a structure further including an external force applying means for applying an external force to the sample to move the sample in the flow path.
With this configuration, the separation accuracy and the time required for the separation can be appropriately set according to the degree to which the external force is applied. Here, it is convenient to use pressure or an electric field as the external force. This is because a large external force applying member is unnecessary. Further, the sample can be moved by utilizing the capillary phenomenon. In this case, the external force applying means is unnecessary, which is advantageous for downsizing the device.

Samples to be separated in the separation apparatus of the present invention include (i) separation and concentration of cells and other components, and (ii) solid components (cell membrane fragments, Separation and concentration of mitochondria, endoplasmic reticulum) and liquid fraction (cytoplasm) (iii) Of the components of the liquid fraction, high molecular weight components (DNA,
Examples include separation of RNA, protein, sugar chain) from low molecular weight components (steroids, glucose, etc.), concentration (iv) separation of polymer decomposition products and undecomposed products. Examples of the microscale include nucleic acids such as nucleic acid fragments, organic molecules such as amino acids, peptides and proteins, metal ions and the like. Of these, the use of nucleic acid or protein as a sample is more effective. When separating these samples, it is necessary to separate small-sized molecules with high resolution, and therefore, a structure provided with minute gaps on the order of several nanometers to several tens of nanometers is essential. On the other hand, it is also required to effectively suppress the clogging caused by huge substances. According to the present invention, both of these requirements can be sufficiently satisfied, and thus it is suitable for separating nucleic acids or proteins.

In the above separating apparatus, a plurality of the sample separating sections formed over the entire cross section of the flow channel may be provided via slits. With such a configuration, the shape of the band in the detection unit becomes linear, the detection region can be expanded, and the detection sensitivity can be improved.

The separating apparatus according to the present invention may be any apparatus as long as it has a sample separating section, and the sample introducing region and the external force applying means may not be provided in the apparatus itself.
For example, the separation device in the present invention may be a disposable cartridge type, and this may be used by incorporating it into a predetermined unit.

Embodiments of the present invention will be further described below with reference to the drawings.

FIG. 1 is a diagram showing an example of a separation apparatus according to the present invention. A separation channel 112 is formed on the substrate 110, and a charging channel 111 and a recovery channel 114 are formed so as to intersect with the separation channel 112. The charging flow channel 111, the separation flow channel 112, and the recovery flow channel 114 have liquid reservoirs 101a, 101b, 102a, and 10 at both ends thereof.
2b, 103a, 103b are formed. The separation channel 112 is provided with a detection unit 113. Appropriate values are selected for the external dimensions of the device depending on the application, but normally, as shown in the figure, length 5 mm to 5 cm, width 3 mm to 3
The value is cm. The sample separation unit is formed in a part of the separation channel 112. The position is appropriately set in consideration of separation efficiency and the like. For example, if the sample is formed on the downstream side of the input channel 111 near the intersection of the input channel 111 and the separation channel 112, the sample can be separated more efficiently.

A method of separating a sample using this apparatus will be described. In the present embodiment, the sample to be separated is usually used in the form of being dissolved or dispersed in pure water, a mixed solution of pure water and a hydrophilic solvent, or a carrier solvent such as a buffer solution.
As the carrier solvent, a mixed solution of water and isopropyl alcohol, an aqueous solution containing trimethylammonium, boric acid and ethylenediaminetetraacetic acid (EDTA), an aqueous solution of sodium phosphate and the like are preferably used.

In the separating operation, first, each channel in the separating device is filled with a carrier solvent. Then, the sample is injected into the liquid reservoir 102a or the liquid reservoir 102b. When injected into the liquid reservoir 102a, a voltage is applied so that the sample flows in the direction of the liquid reservoir 102b, and when injected into the liquid reservoir 102b, a voltage is applied so that the sample flows in the direction of the liquid reservoir 102a. As a result, the sample flows into the charging channel 111, and as a result, fills the entire charging channel 111. At this time, on the separation channel 112, the sample exists only at the intersection with the charging channel 111 and the charging channel 111
Forming a narrow band with a width of about.

Next, the voltage application between the liquid reservoir 102a and the liquid reservoir 102b is stopped, and the liquid reservoir 101a and the liquid reservoir 10 are stopped.
During 1b, a voltage is applied so that the sample flows toward the liquid reservoir 101b. As a result, the sample is separated into the separation channel 112.
During this time, the particles proceed through the separation channel at a speed according to the size of the molecule and the strength of the charge and the size of the gap between the first regions. As a result, different molecular groups in the sample are separated into bands that move at different velocities. These separated bands are detected by the detection unit 11
Up to 3, it is detected by optical or other physicochemical methods. The optical detection is, for example, observing the fluorescence emitted from the molecule by irradiating the detection section 113 with a laser in advance by binding a fluorescent substance to the molecule. The separated bands can be further collected for each band. The voltage application between the liquid reservoir 101a and the liquid reservoir 101b is stopped with reference to the fact that the desired band has passed the detection unit 113, and instead, the liquid reservoir 103a and the liquid reservoir 103b.
Voltage is applied between. Then, in the separation channel 112,
The band existing at the intersection of the recovery channel 114 flows into the recovery channel 114. Liquid reservoir 103a, liquid reservoir 103
When the voltage application between points b is stopped after a certain period of time, desired molecules contained in the separated band are collected in the liquid reservoir 103a or the liquid reservoir 103b.

Further, as shown in FIG. 18, the separation device may be of a type in which the sample is moved by utilizing the capillary phenomenon. In this case, it is not necessary to apply an external force such as electric power or pressure, and energy for driving is unnecessary. In this separation device, a separation flow channel 540 is provided on a substrate 550, an air hole 560 is provided at one end of the separation flow channel 540, and a buffer injection port 510 for injecting a buffer at the time of separation is provided at the other end. Is provided. The separation flow channel 540 is sealed in a portion other than the buffer injection port 510 and the air hole 560. A sample fixed quantity tube 530 is connected to the starting portion of the separation flow path 540, and the sample fixed quantity tube 53
The other end of 0 is provided with a sample injection port 520.

FIG. 20 is an enlarged view showing the vicinity of the sample fixed quantity tube 530. Inside the sample quantification tube 530, sample holding section 503, and buffer introducing section 5
In 04, a hydrophilic absorption region is provided. An absorption region 506 is also provided near the inlet to the separation channel 540. A temporary stop slit 502 is provided between the sample fixed quantity tube 530 and the sample holder 503.
The pause slit 502 can be a hydrophobic area. Pausing slits 505 and 507 separate the absorption regions. The void volume of the sample holding portion 503 is approximately equal to the sum of the void volume of the sample fixed quantity tube 530 and the volume of the temporary stop slit 502. The width of the temporary stop slit 505 is narrower than the width of the temporary stop slit 502. Here, the sample quantification tube 530 has a hydrophilic function and is configured so that it can function as a sample introduction part.

Next, the procedure of the separating operation using the apparatus of FIG. 18 will be described. First, the sample is gradually injected into the sample injection port 520 to fill the sample fixed quantity tube 530.
At this time, make sure that the water surface does not rise. After the sample fixed quantity tube 530 is filled with the sample, the sample gradually exudes into the temporary stop slit 502. When the sample exuding into the temporary stop slit 502 reaches the surface of the sample holder 503, the temporary stop slit 502
And, the sample inside the sample fixed quantity tube 530 is all sucked up by the sample holder 503 having a larger capillary effect. Here, each absorption region is formed so that the degree of hydrophilicity differs depending on the selection of the hydrophilic material,
The sample holder 503 has a larger capillary effect than the sample fixed quantity tube 530. During the filling of the sample holding portion 503 with the sample, temporary stop slits 505 and 507 are provided.
Therefore, the sample does not flow into the buffer introduction part 504.

After the sample is introduced into the sample holder 503, the separation buffer is injected into the buffer injection port 510. The injected buffer is temporarily filled in the buffer introduction part 504, and the interface with the sample holding part 503 becomes linear. When the buffer is further filled, it exudes into the temporary stop slit 505, flows into the sample holding portion 503, and further drags the sample, and advances over the temporary stop slit 507 toward the separation channel. At this time, since the width of the temporary stop slit 502 is larger than the width of the temporary stop slits 505 and 507, even if the buffer flows back to the temporary stop slit 502, the sample has already advanced before the sample holding unit 503. Therefore, there is almost no backflow of the sample.

The separation buffer is a capillary phenomenon and further advances toward the air passage 560 through the separation channel, and in this process, the sample is separated. When the separation buffer reaches the air holes 560, the inflow of the buffer is stopped.
The separation state of the sample is measured when the flow of the buffer is stopped or when the buffer is in progress.

The above embodiment is an example of the separation device using the capillary phenomenon. Another example of sample injection using this principle will be described with reference to FIGS. 19 and 21. In this device, a sample input tube 570 is provided instead of the sample fixed quantity tube 530 in FIG. At both ends of the sample input pipe 570, a sample injection port 520 and
A discharge port 580 is provided.

A separation procedure using this device will be described. First, a sample is put into the sample inlet 520 and the outlet 580 is filled. During this time, the sample
It is absorbed by the sample holder 503 through the charging hole 509.

Thereafter, air is press-fitted into the sample inlet 520 and the sample is discharged from the outlet 580 to wipe the sample in the sample input pipe 570.
dry. In the case of separation by capillarity, a separation buffer is injected in the same manner as above. In case of separation by electrophoresis, the buffer injection port 5
The migration buffer is introduced from the reservoir corresponding to 10 and the reservoir corresponding to the air hole 560. Because of the presence of the widely formed temporary stop slits 505 and 507, the temporary stop slits 505 and 507 do not flow into the sample holder.

At the stage when the sample holding section 503 has finished holding the sample, a small amount of migration buffer is added to the liquid reservoir at one end of the separation channel or the sample holding section 5 is used.
By vibrating lightly around 03, the electrophoretic buffer is made continuous, and a voltage is applied for separation.

Next, the structure of the separation channel in the separation device will be described. FIG. 2 shows the structure of the separation channel 112 or the separation channel 540 in FIG. 1, FIG. 18, or FIG. 19 in detail. In FIG. 2, a groove portion having a depth D is formed in the substrate 701, and the hydrophobic region 70 having a diameter φ is formed in the groove portion.
5 are regularly formed at equal intervals. In this embodiment, the hydrophobic region 705 is formed by attaching or binding a coupling agent having a hydrophobic group to the surface of the substrate 701. Although not shown in FIG. 2, a lid is usually provided on the upper portion of the flow path. This suppresses evaporation of the solvent. Further, it becomes possible to move the sample in the channel by the pressure. However, it is also possible to adopt a structure without a lid.

In FIG. 2, the size of each part is as follows, for example.

W: 10 to 20 micron D: 50 nm to 10 micron Φ: 10 to 1000 nm d: 10 nm to 10 micron p: 50 nm to 10 micron The size of each part is appropriately set according to the purpose of separation. For example, for p, (i) Separation and concentration of cells and other components (ii) Among the components obtained by disrupting cells, solids (fragment of cell membrane, mitochondria, endoplasmic reticulum) and liquid fraction (cytoplasm) ) Separation and concentration (iii) Of the components of the liquid fraction, high molecular weight components (DNA,
In the processing such as separation and concentration of RNA, protein, sugar chain) and low molecular weight components (steroid, glucose, etc.), in the case of (i), 1 μm to 10 μm
m, (ii), 100 nm to 1 μm, (iii)
1 nm to 100 nm. Further, the size of the depth D is an important factor that governs the separation performance, and is preferably about 1 to 10 times, preferably about 1 to 5 times the radius of gyration of the sample to be separated. More preferable.

FIG. 3 is a top view of the structure of FIG.
It is (a)) and a side view (FIG.3 (b)). The hydrophobic region 705 usually has a film thickness of about 0.1 to 100 nm. The surface of the substrate 701 is exposed except for the hydrophobic region 705. By selecting a hydrophilic material such as a glass substrate as the substrate 701, in the structure of FIG. 2, a hydrophobic surface is formed in a predetermined pattern on the hydrophilic surface, and a sample separation function is exhibited. That is, when the above-mentioned hydrophilic buffer solution or the like is used as the carrier solvent, the sample passes only on the hydrophilic surface and does not pass on the hydrophobic surface. Therefore, the hydrophobic region 705 functions as an obstacle for the sample passage, and the sample separation function is exhibited.

Next, the separation method by forming the pattern of the hydrophobic region 705 will be described focusing on the molecular size.
Two methods are mainly considered as the separation method. One is the separation method shown in FIG. In this method, the larger the molecular size, the more the hydrophobic region 705 becomes an obstacle, and the longer it takes to pass through the illustrated separation unit. Those having a small molecular size pass through the gaps between the hydrophobic regions 705 relatively smoothly, and pass through the separation section in a shorter time than those having a large molecular size.

In contrast to FIG. 4, in FIG. 5, large molecules are faster,
It is a method in which small molecules flow out slowly. In the method of FIG. 4, when the sample contains a substance of a huge size, such a substance blocks the interval between the hydrophobic regions 705,
Separation efficiency may decrease. The separation method shown in FIG. 5 solves such a problem. In FIG. 5, a plurality of sample separation sections 706 are formed in the separation channel 112 so as to be separated from each other. In each sample separation unit 706, hydrophobic regions 705 having substantially the same size are arranged at equal intervals.

Since a wide path for allowing large molecules to pass through is provided between the sample separation units 706,
Contrary to Fig. 4, large molecules come out faster and small molecules come out later. This is because as the molecular size becomes smaller, the substance is trapped in the separation region and passes through the longer path, while the larger size substance smoothly passes through the path between the adjacent sample separation units 706. As a result, the small-sized substance is separated after being discharged later than the large-sized substance. Since a substance having a large size passes through the separation region relatively smoothly, the problem that the large molecule is trapped between the hydrophobic regions 705 and the separation efficiency is lowered is reduced, and the separation efficiency is remarkably improved. To be done. In order to make such an effect more remarkable, the width of the path between the adjacent sample separation units 706 is set to the sample separation unit 706.
It is better to make it larger than the gap between the hydrophobic regions 705 inside. The width of the path is preferably about 2 to 200 times, more preferably about 5 to 100 times the gap between the hydrophobic regions 705.

In the example of FIG. 5, the hydrophobic regions 705 having the same size and spacing are formed in each sample separating portion, but the hydrophobic regions 705 having different sizes and spacing are formed in each sample separating portion. You may form.

When a substance having a molecular size is separated, the width of the path between the sample separation parts and the distance between the first regions in the sample separation part are determined by the components (nucleic acid, amino acid, peptide / protein, etc.) to be separated. It is appropriately selected according to the size of the molecule, molecule / ion such as chelated metal ion. For example, the distance between the first regions is preferably the same as or slightly smaller or larger than the radius of gyration of the smallest-sized molecule contained in the sample. Specifically, the difference between the radius of gyration of the smallest molecule contained in the sample and the distance between the first regions is 100 nm or less,
More preferably within 50 nm, most preferably 10 nm
Within By properly setting the interval of the first region, the separation ability is further improved.

Interval between adjacent sample separation parts (width of path)
Is preferably about the same as or slightly smaller or larger than the radius of gyration of the largest-sized molecule contained in the sample. Specifically, the difference between the radius of gyration of the maximum size molecule contained in the sample and the interval between the sample separation portions is within 10%, more preferably within 5%, and most preferably 1% of the radius of gyration of the molecule. Within%. If the space between the sample separation parts is too wide, the separation of small-sized molecules may not be sufficiently performed, and if the space between the sample separation parts is too narrow, clogging may easily occur.

Further, in the above embodiment, the example in which the hydrophobic regions are arranged at a constant interval has been shown, but the hydrophobic regions may be arranged at different intervals in the sample separation section. By doing so, it is possible to efficiently separate large / medium / small molecules / ions of a plurality of sizes. Regarding the arrangement of the hydrophobic regions, it is also effective to adopt a method of arranging the hydrophobic regions alternately with respect to the traveling direction of the sample. By doing so, the target component can be efficiently separated.

In the separation apparatus of the present invention, as shown in FIG. 6, a voltage is applied to both ends of the separation channel 112, whereby the sample moves in the separation channel 112. Here, in addition to the voltage for applying an external force to the sample, a voltage for suppressing the electroosmotic flow may be applied. In the configuration of FIG. 6, a zeta correction voltage is applied to the substrate for this purpose.
In this way, the electroosmotic flow is suppressed and the broadening of the measurement peak can be effectively prevented.

Next, a method of manufacturing the separation device shown in FIG. 13 will be described with reference to the drawings.

The separation device of FIG. 13 has a structure in which the separation / recovery channel is omitted from the separation device of FIG. This device does not aim to separate the separated substances of the sample, but analyzes the components separated by the detection unit 113. A sample separation region is provided in the separation channel 112. The surface of the sample separation region is composed of a plurality of hydrophobic regions arranged two-dimensionally at substantially equal intervals and a hydrophilic region occupying the surface of the sample separation portion excluding the hydrophobic region.

In the separation device of FIG. 13, first, as shown in FIG. 7A, a groove portion 730 is provided on the surface of the substrate 701, and then, as shown in FIG. 7B, sample separation is performed at a predetermined position in the groove portion 730. Obtained by forming the region 731. Less than,
A process of forming the groove portion 730 on the substrate 701 of FIG. 7A will be described with reference to FIG. Note that in this embodiment, an example in which a glass substrate is used as the substrate 701 will be described.

First, the hard mask 71 is formed on the substrate 701.
0 and a resist mask 711 are sequentially formed (FIG. 8).
(A)). Then, a predetermined opening is provided in the resist mask 711 (FIG. 8B). Then, dry etching is performed using the resist mask 711 provided with the opening as a mask to obtain the state of FIG. SF ₆ or the like is used as the etching gas. Subsequently, the substrate 701 is wet-etched using an etching solution such as buffered hydrofluoric acid. Usually, the etching depth is about 1 μm. FIG. 8D shows a state in which this etching is completed. Finally, the hard mask 710 and the resist mask 71
1 is removed (FIG. 8 (e)). Through the above steps, FIG.
A groove portion 730 as shown in (a) is formed.

In the step of forming the groove portion 730 in FIG. 7A, the surface of the groove portion 730 can be made hydrophilic and the other surface of the substrate 701 can be made hydrophobic. Less than,
A process of forming such a structure will be described with reference to FIG. First, a hydrophobic surface treatment film 720 is formed on the entire surface of the structure obtained in FIG. 8E (FIG. 9A).
Examples of the material forming the hydrophobic surface treatment film 720 include 3-thiolpropyltriethoxysilane.

Subsequently, a resist 721 is applied to the surface of the substrate by spin coating and dried (FIG. 9B). Next, an opening is provided in the resist 721 corresponding to the groove (FIG. 9).
(C)). Next, dry etching is performed using the resist 721 provided with an opening as a mask (FIG. 9D). After that, the resist 721 is removed by ashing and stripping solution treatment. By performing the above steps, FIG.
The state of (e) is obtained. That is, the inner wall of the sample flow channel is
The hydrophilic surface of the substrate 701 made of a glass material is exposed, while the other portion is covered with the hydrophobic surface treatment film 720. Therefore, if a hydrophilic solvent is used as the carrier solvent, the sample will not flow out of the groove.

Next, the process of forming the sample separation region 731 in FIG. 7B will be described with reference to FIG.
First, as shown in FIG. 10A, an electron beam exposure resist 702 is formed on a substrate 701. Then, the electron beam exposure resist 702 is pattern-exposed into a predetermined shape using an electron beam (FIG. 10B). When the exposed portion is dissolved and removed, an opening patterned into a predetermined shape is formed as shown in FIG. After that, FIG.
Oxygen plasma ashing is performed as shown in (d). In addition,
Oxygen plasma ashing is necessary when forming a submicron-order pattern. This is because if oxygen plasma ashing is performed, the base to which the coupling agent adheres is activated and a surface suitable for precise pattern formation is obtained. On the other hand, it is less necessary when forming a large pattern on the order of microns or more.

After the ashing is completed, the state shown in FIG. In the figure, the hydrophilic region 703 is formed by depositing resist residues and contaminants. In this state, the hydrophobic area 705 is formed (FIG. 11B). As a method for forming the film forming the hydrophobic region 705, for example, a vapor phase method can be used. In this case, a film is formed by placing a substrate and a liquid containing a coupling agent having a hydrophobic group in a closed container and leaving it for a predetermined time. According to this method, since the solvent or the like does not adhere to the surface of the substrate, it is possible to obtain a processed film having a desired precise pattern. A spin coating method can also be used as another film forming method. In this case, a coupling agent solution having a hydrophobic group is applied and surface treatment is performed to form the hydrophobic region 705. As the coupling agent having a hydrophobic group, 3-thiolpropyltriethoxysilane can be used. As a film forming method, a dipping method or the like can also be used. The hydrophobic region 705 does not deposit on the hydrophilic region 703,
Since it is deposited only on the exposed portion of the substrate 701, a surface structure in which a large number of hydrophobic regions 705 are formed at intervals is obtained as shown in FIG.

In addition to the process described above, the same surface structure as above can be obtained by the following method. According to this method, after the unexposed portion 702a patterned as shown in FIG. 10C is formed, the oxygen plasma ashing is not performed, and the resist opening 3 is formed as shown in FIG.
Deposit thiolpropyltriethoxysilane to form hydrophobic regions 705. After that, wet etching is performed using a solvent that can selectively remove the unexposed portion 702a to obtain the structure of FIG. At this time, it is important to select a solvent that does not damage the film forming the hydrophobic region 705. Examples of such a solvent include acetone and the like.

In the above-mentioned embodiment, the hydrophobic region is formed in the flow channel groove, but the following method can be adopted instead. First, two types of substrates are prepared as shown in FIGS. 14 (a) and 14 (b). The substrate of FIG. 14A has a structure in which a hydrophobic film 903 made of a compound having a hydrophobic group such as 3-thiolpropyltriethoxysilane is formed on a glass substrate 901. The hydrophobic film 903 is formed in a predetermined patterning shape. This hydrophobic film formation 90
The place where 3 is provided becomes the sample separation part. On the other hand, FIG.
The substrate (b) has a structure in which a stripe-shaped groove is provided on the surface of the glass substrate 902. This groove portion serves as a sample flow path. The method for forming the hydrophobic film 903 is as described above. As described above, the stripe-shaped groove on the surface of the glass substrate 902 can be easily formed by wet etching using a mask. These are shown in FIG.
The sample separation device according to the present invention can be obtained by laminating as described above. A space 904 formed by the two substrates serves as a sample channel. According to this method, since the hydrophobic film 903 is formed on the flat surface, the manufacturing is easy and the manufacturing stability is good.

As a method for producing the coupling agent film, "N
ATURE, vol.403, 13, January (2000) ", a hydrophilic / hydrophobic micropattern is formed by forming a film of a silane coupling agent on the entire surface of the substrate by the LB film pulling method. Can be formed.

Further, in the present invention, the sample separation area may be provided with only one hydrophobic area. In this case, for example, one hydrophobic region extending in the flow direction of the sample can be formed in the separation channel having a hydrophilic surface. Also in this case, when the sample passes through the separation channel, the sample can be separated by the surface characteristics of the sample separation region.

Further, the flow path itself can be formed by the above-mentioned hydrophobic treatment and hydrophilic treatment.

When the channel is formed by the hydrophobic treatment, a hydrophilic substrate such as a glass substrate is used to form a portion corresponding to the wall of the channel in the hydrophobic region. Since water enters while avoiding the hydrophobic region, a flow path is formed between the wall portions. The channel may or may not be covered with a lid, but when the lid is covered, it is preferable to leave a gap of several μm from the substrate. For the gap, use the PDMS (polyd
This can be achieved by bonding viscous resin such as imethylsiloxane) or PMMA to the substrate as glue. Even if the adhesive is adhered only near the stump, when the water is introduced, the hydrophobic region repels the water, so that the flow path is formed.

On the other hand, when the channel is formed by the hydrophilic treatment, the hydrophilic channel is formed on the surface of the hydrophobic substrate or the surface of the substrate which is made hydrophobic by the silazane treatment or the like. Also in this case,
Since water enters only the hydrophilic region, the hydrophilic region can be used as a flow path.

Further, this hydrophobic treatment or hydrophilic treatment can be carried out by using a printing technique such as a stamp or ink jet. The stamping method uses PDMS resin. The PDMS resin is polymerized with silicone oil to be made into a resin, but even after the resin is made into a resin, the gaps between the molecules are still filled with the silicone oil. Therefore, when the PDMS resin is brought into contact with a hydrophilic surface, for example, a glass surface,
The contacted part becomes strongly hydrophobic and repels water. Using this, PDMS with recesses formed at the positions corresponding to the flow path
By using the block as a stamp and bringing it into contact with the hydrophilic substrate, the flow path by the above-mentioned hydrophobic treatment can be easily manufactured.

In the ink jet printing method,
A low-viscosity type silicone oil is used as an ink for inkjet printing, and a hydrophilic resin thin film such as polyethylene, PET, cellulose acetate, or a cellulose thin film (cellophane) is used as printing paper. The same effect can be obtained by printing in a pattern such that silicone oil adheres to the flow path wall portion.

Further, a hydrophobic patch or a hydrophilic patch having a predetermined shape is formed by the hydrophobic treatment and the hydrophilic treatment, and a substance having a size smaller than a specific size is allowed to pass therethrough and a substance having a size larger than the specific size is not passed through the filter. Can also be formed in the flow path.

For example, when a filter is composed of hydrophobic patches, a dotted filter pattern can be obtained by arranging the patches linearly and repeatedly at regular intervals. The distance between the hydrophobic patches should be larger than the size of the substance you want to pass through and smaller than the size of the substance you do not want to pass. For example, when you want to remove a substance of 100 μm or more, the distance between the hydrophobic patches is
The width is set to be narrower than 100 μm, for example, 50 μm.

The filter can be realized by integrally forming the hydrophobic region pattern for forming the flow path and the pattern of the hydrophobic patch formed in the broken line shape. As the forming method, the above-mentioned method by photolithography and SAM film formation, stamping method, inkjet method, or the like can be appropriately used.

When a filter is constructed in the flow path,
The filter surface may be provided perpendicularly to the flow direction, or the filter surface may be provided parallel to the flow direction. When the filter surface is provided in parallel to the flow direction, there is an advantage that the substance is less likely to be clogged and the area of the filter can be made larger than when it is provided vertically. In this case, the width of the flow path portion is made wider, for example, 1000 μm, and 50 μ
mx 50 μm square hydrophobic patches are
By forming in the flow direction of the flow path so as to have a gap of m, the flow path can be divided into two parallel to the flow direction. When a liquid containing a substance to be separated is introduced from one side of the divided channels, the filtrate from which substances larger than 50 μm contained in the liquid are removed flows out to the other channel. This allows the substance to be concentrated on one side of the flow path.

[0090]

Example A separation channel was prepared as follows. First, a microscope cover glass (24 mm × 50 mm) was used as a substrate, and a separation region (width 10 mm × length 50 mm) extending in the longitudinal direction was formed in the central portion of the substrate. The separation region was formed by arranging 100 μm × 100 μm square hydrophobic patches in a square lattice so that the gap between the patches was 200 μm.

The hydrophobic patch was formed as follows. First, on the cover glass, 100 μm × 10
A square of 0 μm was exposed by ordinary photolithography using a negative resist (S1818), and the resist in the square portion was removed by development. Subsequently, the surface was subjected to oxygen plasma ashing (350 W, 0.5 Torr, 10 minutes), and then a hydrophobic silazane SAM film (self-assembled monolayer) was formed on the exposed cover glass surface using silazane vapor. Then, the resist was removed with acetone.

Two cover glasses having the hydrophobic patch thus formed were prepared. One of the cover glasses was attached to a slide glass of 10 cm × 10 cm with an instant adhesive, and the thickness of the cover glass was 18 μm in the area other than the separation area.
Then, the polyethylene sheet was placed. Subsequently, another cover glass was placed on the polyethylene sheet so that the treated surfaces face each other to form a flow path.

1 × TBE buffer was introduced by a pipette into one end of the separation channel (width 10 mm × length 50 mm × depth 18 μm) thus formed. 1 x TB
The E buffer was automatically filled in the separation channel by the capillary effect.

FIG. 16 is a micrograph showing the pattern of bubbles formed after introducing 1 × TBE buffer. It can be seen that round bubbles are formed at the positions where the hydrophobic patches are formed, and bubbles are formed in the cross section of the flow channel. The distance between the bubbles was about 300μ.

Next, a substance having a size similar to that of a general cell was used to perform separation using the separation channel. Typical cell sizes are about 1 μm to 10 μm. In particular, red blood cells have a diameter of 7.5 μm, white blood cells have a diameter of 10 μm, platelets have a diameter of 2 μm, and bacteria have a diameter of 1 μm. In this embodiment,
Two kinds of fluorescent beads of Φ1 μm and Φ10 μm (Fluoresbright Carboxylate (2.
5% Solid-Latex)) was used.

Subsequently, the above-mentioned two types of fluorescent beads were 1 ×
A bead suspension is appropriately diluted with TBE buffer to a concentration suitable for observation, and the bead suspension is added to one end of the separation channel filled with 1 × TBE buffer with 0.50 μ
l was added dropwise. The dropped bead suspension entered and stopped in the flow channel by the same capillary phenomenon. When observed under a microscope, the two types of beads could be clearly distinguished by the difference in size.

Next, 200 μl was added to the same end of the separation channel.
1 × TBE buffer was dropped at once. The dropped buffer automatically entered the separation channel and overflowed from the other end. In the process, the bead suspension at the end of the channel was pushed through the separation channel and moved in the separation channel. The movement of the bead suspension was observed with a CCD camera.

When 1 × TBE buffer was added and time passed, beads with a diameter of 10 μm sank on the bottom of the flow channel. Therefore, observation was performed while beads were floating for 3 seconds after the addition.

FIG. 17 shows how the beads collide.
The beads are moving from the right to the left in the figure at about 300 μm / sec. Both types of beads were moving at almost the same speed on the flow except the bubbles, but both stopped temporarily when they collided with the bubbles on the hydrophobic patch. After that, the bubbles flowed around, but the moving speed thereof was reduced to about one-third of the flow velocity of the portion other than the bubbles. This shows that the movement of the beads is hindered by the hydrophobic patch and the bubbles formed on it. The speed at which the beads wrap around the air bubbles is obviously slower for the larger size φ10 μm beads (indicated by 1 in the figure), and is smaller in the smaller size φ1 μm beads (indicated by 2 in the figure, where shutter speed relationship is used). It appears to be streaky). This shows the separation effect that the collision with the hydrophobic patch causes a velocity difference due to the size of the beads. It is considered that the separation effect becomes more remarkable if the collision frequency of the beads is increased with the hydrophobic patch as a more dense pattern.

From the results of the above examples, the following was shown. (I) Bubbles are formed on the patch (hydrophobic patch) by the hydrophobic surface treatment. (Ii) The beads cannot enter inside the bubbles on the patch, and the bubbles on the hydrophobic patch act as an obstacle in the flow path. (Iii) The moving speed of both types of beads having different sizes decreases due to contact with the bubbles on the hydrophobic patch. (Iv) The moving speed of beads differs depending on the size. (Large beads are slower than the moving speed of smaller beads.) Therefore, the beads can be separated due to the difference in size.

[0101]

As described above, according to the present invention, the pattern of the hydrophilic region and the hydrophobic region is formed on the surface of the sample separating portion, and the sample is separated by the surface characteristics. That is, the hydrophilic region or the hydrophobic region formed separately on the surface of the sample separation portion has a function as a sieve, and thereby the target component is efficiently separated. At this time, since the sample is separated depending on the size and polarity of the sample, it is possible to realize an excellent separation performance that has never been obtained. Further, since the sample separation portion can be formed by surface processing, the manufacturing stability is excellent. In addition, because the separation is done by the surface characteristics, a small amount of sample
The time required for separation is also short. Further, the problem of clogging can be solved, and after use, the surface of the sample separation part can be washed very easily by a method such as flowing a washing liquid. Therefore, both high-precision separation characteristics and excellent operability are realized.

[Brief description of drawings]

FIG. 1 is a diagram showing an example of a separation device according to the present invention.

FIG. 2 is a diagram showing in detail a structure of a separation channel in FIG.

FIG. 3 is a diagram showing in detail the structure of a separation channel in FIG.

FIG. 4 is a diagram for explaining a sample separation method.

FIG. 5 is a diagram for explaining a sample separation method.

FIG. 6 is a diagram showing a method of applying a correction voltage for adjusting the electroosmotic flow.

FIG. 7 is a plan view showing a schematic structure of a separation device according to the present invention.

FIG. 8 is a process cross-sectional view for explaining the method for manufacturing the separation device according to the present invention.

FIG. 9 is a process cross-sectional view for explaining the method for manufacturing the separation device according to the present invention.

FIG. 10 is a process sectional view for explaining the manufacturing method for the separation device according to the present invention.

FIG. 11 is a process sectional view for explaining the manufacturing method for the separation device according to the present invention.

FIG. 12 is a process sectional view for explaining the manufacturing method for the separation device according to the present invention.

FIG. 13 is a plan view showing a schematic structure of a separation device according to the present invention.

FIG. 14 is a drawing for explaining the manufacturing method of the separation device according to the present invention.

FIG. 15 is a sectional view showing a schematic structure of a separation device according to the present invention.

FIG. 16 is a micrograph showing a pattern of bubbles formed at positions where hydrophobic patches are formed in the separation channel.

FIG. 17 is a diagram showing how beads collide with each other in a separation channel.

FIG. 18 is a diagram showing another example of the separation device.

FIG. 19 is a diagram showing another example of the separation device.

20 is an enlarged view of the vicinity of the sample quantification tube of the separation device shown in FIG.

FIG. 21 is a detailed view of the separation device shown in FIG.

[Explanation of symbols]

101a, b liquid reservoir 102a, b liquid reservoir 103a, b liquid reservoir 110 substrate 111 Input flow path 112 Separation flow path 113 detector 114 recovery channel 502 Suspended slit 503 sample holder 504 buffer introduction section 505 pause slit 506 Absorption area 507 Pause slit 509 input hole 510 buffer inlet 520 sample inlet 530 sample determination tube 540 Separation flow path 550 substrate 560 air holes 570 sample input tube 580 outlet 701 substrate 702 Electron beam exposure resist 702a Unexposed part 702b Exposure unit 703 hydrophilic region 705 hydrophobic region 706 Sample separation unit 710 hard mask 711 resist mask 720 Hydrophobic surface treatment film 721 resist 730 groove 731 Sample separation area 902 glass substrate 903 hydrophobic membrane 904 space

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 37/00 101 G01N 27/26 331Z 331E (72) Inventor Hisao Kawaura 5-7 Shiba, Minato-ku, Tokyo No. 1 Inside the NEC Corporation (72) Inventor Toru Sano No. 5-7 Shiba, Minato-ku, Tokyo No. 1 Inside the NEC Corporation (72) Riji Sakamoto 5-7-1 Shiba, Minato-ku, Tokyo No. Nippon Electric Co., Ltd. (72) Inventor Noriyuki Iguchi 5-7 Shiba, Minato-ku, Tokyo Nippon Electric Co., Ltd. (72) Inventor Hiroko Someya 5-7-1 Shiba, Minato-ku, Tokyo Japan Electric stock company

Claims (24)

[Claims] 1. A separation device comprising a substrate, a flow path formed on the surface of the substrate for a sample to pass through, and a sample introduction section and a sample separation section provided in the flow path, wherein the sample separation section The surface of the separator has a hydrophilic region and a hydrophobic region. 2. A substrate, a channel formed on the surface of the substrate through which a sample passes, a sample introducing section and a sample discharging section provided in the channel, and from the sample introducing section to the sample discharging section. A separation device comprising a sample separation part provided in a flow path between, wherein the surface of the sample separation part excludes a plurality of first regions arranged apart from each other. A second region occupying the surface of the sample separation part, wherein one of the first region and the second region is a hydrophobic region and the other is a hydrophilic region. . 3. The separation device according to claim 2, wherein the first regions are two-dimensionally arranged at substantially equal intervals. 4. The separation apparatus according to claim 2 or 3, wherein a plurality of the sample separation units are provided. 5. The separation device according to claim 4, wherein the interval between the adjacent sample separation parts is wider than the interval between the first regions forming each sample separation part. 6. The separation apparatus according to claim 4 or 5, wherein the intervals of the first regions in the respective sample separation units are different from each other. 7. The separation apparatus according to claim 1, further comprising an external force applying means, wherein the sample is moved by the external force from the sample introducing section toward the sample separating section. And a separation device. 8. The separation device according to claim 1, further comprising a detection unit downstream of the sample separation unit. 9. The separation device according to claim 1, wherein the hydrophobic region is composed of a membrane containing a compound having a hydrophobic group. 10. The separation device according to claim 9, wherein
The separation device, wherein the compound having a hydrophobic group is a silane coupling agent having a hydrophobic group. 11. The separation device according to claim 9, wherein the hydrophobic group is a thiol group. 12. The separation device according to claim 1, wherein the hydrophobic region contains a silicone compound. 13. The separation device according to claim 1, wherein the hydrophilic region is composed of a membrane containing a compound having a hydrophilic group. 14. The separation device according to claim 13, wherein the compound having a hydrophilic group is a silane coupling agent having a hydrophilic group. 15. The separation device according to claim 14, wherein the silane coupling agent is a compound having an amino group. 16. A sample separation method, which comprises using the separation apparatus according to claim 1 to introduce a sample from the sample introduction unit to separate a predetermined component in the sample. 17. A method of manufacturing a separation device, comprising: a substrate; a flow path formed on the surface of the substrate for a sample to pass through; and a sample separation section provided in the flow path, the method comprising: A step of forming the channel having, and at least a part of the surface of the channel, after providing a mask having an opening, depositing a compound having a hydrophobic group from the opening to the surface of the channel, then And a step of forming the sample separation part in which the hydrophobic region is arranged by removing the mask, the manufacturing method of the separation device. 18. The method for producing a separation device according to claim 17, wherein the compound is a silane coupling agent having a hydrophobic group. 19. The method for manufacturing a separation device according to claim 18, wherein the hydrophobic group is a thiol group. 20. A method of manufacturing a separation device comprising a substrate, a flow path formed on the surface of the substrate for a sample to pass through, and a sample separation section provided in the flow path, the method comprising: A step of forming the channel in the substrate having, and after providing a mask having an opening on at least a part of the surface of the channel, depositing a compound having a hydrophilic group from the opening to the surface of the channel, Next, the step of removing the mask to form the sample separating section in which the hydrophilic region is arranged is included. 21. The method for producing a separation device according to claim 20, wherein the compound is a silane coupling agent having a hydrophilic group. 22. The method for manufacturing a separation device according to claim 21, wherein the hydrophilic group is an amino group. 23. A method for manufacturing a separation device, comprising: a substrate; a flow path formed on the surface of the substrate for a sample to pass through; and a sample separation unit provided in the flow path, the method comprising: A step of forming the channel having the step of forming a sample separation section in which a liquid silicone compound is attached to the surface of the channel and a hydrophobic region is arranged. Device manufacturing method. 24. The method for manufacturing a separation device according to claim 23, wherein in the step of forming the sample separation portion, a surface of a silicone resin containing the liquid silicone compound is brought into contact with the flow channel surface. A method for manufacturing a separation device, comprising: