JP2005249739A - Electroendosmotic flow cell, insulator sheet, and electroendosmotic flow cell chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To generate a unidirectional electroendomsmotic flow. <P>SOLUTION: In this electroendomsmotic flow cell, at least a bottom face of a flow passage for the electroendomsmotic flow comprises a charged material or a material charge-treated artificially. The flow passage comprises a uniform width of central part, and two end parts serving as the start point and the final point of the flow passage, the central part is continuous from a side part side to the respective end parts, as to its longitudinal direction. The two end parts of a hollow part may be located in the same side or a different side as to the longitudinal direction of the central part, in the electroendomsmotic flow cell. The two flow passage may be formed into a fylfot shape of a crossed type thereof, and the central part may be located nonlinearly between the two end parts. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a flow cell, apparatus and process using electroosmotic flow.

  The electroosmotic flow (EOF) is a flow generated by ions in the electric double layer generated at the solid / liquid interface moving by an electric field applied from the outside. In the case of a glass substrate, when the pH of the solution in contact with the glass substrate is 3 or more, the surface is negatively charged due to dissociation of the silanol groups on the surface, and cations in the solution are attracted to the surface to form an electric double layer. When a DC electric field is applied to the electric double layer, an electroosmotic flow due to cations flows, and a strong shear flow is generated near the substrate.

Electroosmotic flow is expected as an alternative to traditional water pressure driven microfluidic devices [Stroock, AD, et al., Physical Review Letters 84 (15), 3314 (2000)]. This is because a precise pump is required for pressure driving, but a simple anode / cathode electrode pair and a charged surface are required for electroosmotic flow, and electroosmotic flow is difficult to obtain by pressure driving. This is because a shearing force can be generated. The superiority of these electroosmotic flows is indispensable for the realization of a small chemical analyzer (mTAS, micro Total Analysis system, or portable medical diagnostic device). For example, as part of the development of a genome analysis and modification device, Washizu et al. Attempted to expand (and convert to fiber) long DNA with a round shape into a straight line by using electroosmotic flow. [Washizu et al., Journal of Electrostatics. 57 (3), 395 (2003)].
Stroock, AD, et al., Physical Review Letters 84 (15), 3314 (2000) Washizu et al., Journal of Electrostatics. 57 (3), 395 (2003)

  As mentioned earlier, Washizu et al. Attempted to extend and expand long-chain DNA linearly using electroosmotic flow. However, a uniform direction of electroosmotic flow, which is indispensable for the transportation of cells that are nested DNA and the fiber formation of DNA by shearing force, has not yet been achieved with good reproducibility.

  An object of the present invention is to generate a uniformly oriented electroosmotic flow.

  A first electroosmotic flow cell according to the present invention is an electroosmotic flow cell in which an electroosmotic flow channel is formed, and is made of a material having at least a bottom surface of the flow channel or an artificially charged material. The flow path is composed of a central portion having a uniform width and two end portions which are a start point and an end point of the flow path, and the central portion continues from the side portion side to the end portions in the longitudinal direction. . In the electroosmotic flow cell, the two end portions of the hollow portion may be on the same side or different sides with respect to the longitudinal direction of the central portion.

  In the first electroosmotic flow cell, for example, a plurality of the flow paths are provided, and the central portions of the plurality of flow paths cross each other.

  Further, in the first electroosmotic flow cell, for example, a plurality of the flow paths are provided, and the central portions of the plurality of flow paths are provided with common end portions.

  The second electroosmotic flow cell according to the present invention is an electroosmotic flow cell having an electroosmotic flow channel, and at least the bottom surface of the flow channel is made of a charged material or an artificially charged material. The flow path includes a central portion having a uniform width and two end portions serving as a start point and an end point of the flow path, and the central portion exists in a non-linear shape between the two end portions.

  In the second electroosmotic flow cell, preferably, one or more columnar portions are further provided on the bottom surface and in the flow path.

  In the second electroosmotic flow cell, preferably, one or more wall-like portions are further provided on the bottom surface and in the hollow portion of the insulator sheet.

  The first or second electroosmotic flow cell includes, for example, a substrate made of a charged material or an artificially charged material, and an insulator sheet placed on the surface of the substrate. The flow path is formed as a hollow portion of the insulator sheet. Alternatively, the entire flow cell may be made of a charged material (excluding electrodes) or an artificially charged material.

  In the first or second electroosmotic flow cell, preferably, an electrode is further provided at each of the two ends.

  The first or second electroosmotic flow cell preferably further includes an inlet for injecting the sample into the channel and an outlet for discharging the sample from the channel. Further, an injection pump for injecting the sample into the injection port and a discharge pump for discharging the sample from the discharge port may be provided.

  An electroosmotic flow cell device according to the present invention includes the first or second electroosmotic flow cell described above and a constant voltage power source that applies a DC constant voltage to electrodes provided at each end of the electroosmotic flow cell.

  The first insulator sheet according to the present invention (for example, a silicon rubber sheet) is installed on the surface of a substrate made of a charged material or an artificially charged material to form an electroosmotic flow cell. It is a body sheet, and has a hollow portion composed of a central portion having a uniform width to be a flow channel and two end portions to be a start point and an end point of the flow channel. The central portion continues to each of the end portions from the side portion side in the longitudinal direction.

  The second insulator sheet according to the present invention (for example, a silicon rubber sheet) is an insulator sheet for forming an electroosmotic flow cell installed on the surface of a charged material or an artificially charged material. In addition, a hollow portion including a central portion having a uniform width to be a flow path and two end portions to be a start point and an end point of the flow path is provided. The central part exists in a non-linear shape between the two ends.

  A first electroosmotic flow cell chip according to the present invention comprises a substrate made of a charged material or an artificially charged material, and an insulator layer placed on the surface of the substrate, The insulator layer includes a plurality of hollow portions in a matrix. Here, each hollow portion is composed of a central portion having a uniform width serving as a transport channel and two end portions serving as a start point and an end point of the transport channel. It continues to each said edge part from the part side.

  A second electroosmotic flow cell chip according to the present invention comprises a substrate made of a charged material or an artificially charged material, and an insulator layer formed on the surface of the substrate. The insulator layer includes a plurality of hollow portions in a matrix shape, and the shape of each hollow portion includes a central portion having a uniform width serving as a transport channel, and two end portions serving as a start point and an end point of the transport channel. And the central portion exists in a non-linear shape between both end portions.

  In the first or second electroosmotic flow cell chip, preferably, electrodes are further provided at both ends of the hollow portion.

  In the analysis method according to the present invention, an electroosmotic flow cell having a channel formed on a charged material or an artificially charged material is prepared, and one end of DNA or a linear polymer is fixed to the channel. Then, the liquid is introduced into the flow path. Next, by applying a constant voltage to the electrodes of the electric flow cell to generate an electroosmotic flow that flows in a uniform direction, the DNA or linear polymer is developed and aligned in the direction of the uniform electroosmotic flow. To analyze.

  In the transport method according to the present invention, an electroosmotic flow cell having a flow path formed on a charged material or an artificially charged material is prepared, and a liquid containing molecules having a size of 10 nm to 100 μm is flowed. To introduce. Next, a constant voltage is applied to the electrodes of the electroosmotic flow cell to generate an electroosmotic flow that flows in a uniform direction, thereby transporting molecules having a size of 10 nm to 100 μm.

In the treatment method according to the present invention, an electroosmotic flow cell having a flow path formed on a charged material or an artificially charged material and having a plurality of columnar portions formed in the flow path is prepared. One end of the linear polymer is fixed to the channel, and the liquid is introduced into the channel. Next, a constant voltage is applied to the electrodes of the electroosmotic flow cell to generate an electroosmotic flow that flows in a uniform direction, and the DNA or linear polymer is developed and aligned in the direction of the uniform electroosmotic flow, By changing the voltage applied to the electrode to change the direction of the applied electric field to change the direction of the electroosmotic flow, the developed DNA or linear polymer is suspended between the columnar parts.
In addition, the component demonstrated above of this invention can be combined as much as possible.

The electroosmotic flow cell according to the present invention can generate an electroosmotic flow in a uniform direction.
The electroosmotic flow cell according to the present invention can be used as a transfer device (actuator) for proteins, cells (blood) and the like.
The electroosmotic flow cell according to the present invention can be used for DNA manipulation (elongation, etc.) and modification.
The electroosmotic flow cell according to the present invention can be incorporated in a microfluidic device such as a micro transport device or a small chemical analyzer.

  Embodiments of the present invention will be described below with reference to the accompanying drawings. In the drawings, the same reference symbols denote the same or equivalent. The embodiments described below do not limit the invention described in the claims.

  FIG. 1 shows an outline of an electron osmotic flow cell apparatus. The electroosmotic flow cell includes, for example, a glass substrate 1 and an insulator sheet 2 (for example, a rubber plate made of silicon rubber) installed on the surface of the glass substrate. In order to introduce a liquid sample into the insulator sheet 2. The hollow portion 3 is formed as a flow path. The hollow part (flow path) for generating the electroosmotic flow is created by cutting a silicon rubber plate, for example. By attaching the rubber plate to a glass substrate or the like, a cut portion provided in advance in the rubber plate functions as a flow path. The hollow part 3 becomes a chamber for introducing a liquid sample. The glass substrate 1 is transparent and suitable for observation with a microscope. The glass substrate is an example of a substrate made of a charged material or an artificially charged material, and a material other than glass (for example, a naphthoin film or plasma) is used to generate an electroosmotic flow by an electric double layer. Ashing silicon) may be used.

  When using this apparatus, when a liquid sample is introduced into the hollow portion 3, the surface of the glass substrate 1 on the lower side of the solution is negatively charged, and the cations in the solution are attracted to the solid / liquid interface. An electric double layer is formed. Now, when a DC voltage is applied between a pair of electrodes (for example, platinum foil) provided at both ends of the flow path by the DC constant voltage power supply device 4, the cations in the electric double layer are moved by the electric field, Generates osmotic flow. A battery may be used in place of the DC constant voltage power supply device 4.

  In the present invention, as described below, hollow portions or flow paths having various shapes are used in the electron permeation flow cell. In the flow cell shown in FIG. 1 and FIG. 2 (a U-shaped (cis-type) flow cell), the hollow portion 3 is a rectangular central portion 11 having a uniform width to be a flow path, and a start point and an end point of the flow path. It consists of two, for example, square ends 12,13. The central portion 11 communicates with the end portions 12 and 13 from the side portion side in the longitudinal direction. The two end portions 12 and 13 are on the same side with respect to the longitudinal direction of the central portion 11. The direction of the electric field is uniform at the center. The hatched portion indicates an example of the position of the electrode 5 provided at the end. Moreover, the sign of + and-in a figure shows the polarity of an applied voltage, and the arrow in the center part 11 shows the direction through which the cation flows in an electric double layer.

  Further, the flow cell (transformer type flow cell) shown in FIG. 3 is different from the structure shown in FIG. 2 in that the two end portions 12 ′ and 13 ′ are different in the longitudinal direction of the central portion 11 (opposite side). Is to be located. The flow cell shown in FIG. 4 is a modification of the structure shown in FIG. 2, and the two end portions 12 ″ and 13 ″ are elongated semicircular connecting portions connecting the circular portion and the central portion 11. It consists of. In the structure shown in FIGS. 2 to 4, the electroosmotic flow flows from the positive electrode to the negative electrode. As will be explained later, this simple new structure ensures a uniform electroosmotic flow.

  The flow cell (saddle type flow cell) shown in FIG. 5 is a combination of two transformer type flow cells (FIG. 3) so as to cross each other at the center. The two central portions 21a and 21b are orthogonal to each other, the end portions 22a and 23a are connected to the central portion 21a, and the end portions 22b and 23b are connected to the central portion 21b. Further, two pairs of electrodes are provided. In this vertical flow cell, two orthogonal electric fields can be applied. In the figure, a + and a− indicate the polarity of the voltage applied to the first flow path, and b + and b− indicate the polarity of the voltage applied to the second flow path. Further, in the flow cell shown in FIG. 6, the structures of the end portions 22a ', 22b', 23a ', and 23b' include a circular portion and an elongated connecting portion that connects the central portion 21a 'and 21b'. In these flow cells, two electric fields generated by two pairs of electrodes can be combined to continuously change the direction of flow. For example, as shown on the lower side of FIG. 5, the electroosmotic flow can be made to flow in eight directions including an oblique direction. Thereby, a shearing force can be applied to the substance from multiple directions, and the substance can be conveyed in multiple directions. The fluid circuit required for a small chemical analyzer or the like is usually composed of a combination of two intersecting flow paths. However, the multi-channel flow cell shown in FIG. 5 is required for a small chemical analyzer or the like. Can be used in such fluid circuits.

  In the example shown in FIG. 7, the central portion 31 has a curved shape with a constant width. In this example, the central portion 31 has a shape in which two semicircles are combined, and circular end portions 32 and 33 continue to both ends of the central portion 31. Since the central portion 31 is curved, the electroosmotic flow flows non-linearly.

  Moreover, in the example shown in FIG. 8, although the some flow path is provided, the both ends are connected. The two flow paths are composed of a central portion 41a, 41b and end portions 42a, 42b, 43a, 43b continuing on both sides thereof, and the end portions 42a, 42b and 42a, 43b are connected to each other. That is, the plurality of flow paths have a common end. Thus, when a voltage is applied between the pair of electrodes, a uniform electroosmotic flow is generated at the two central portions 41a and 41b. Note that the number of flow paths is not limited to two.

  In the example shown in FIG. 9, in the flow cell having the structure shown in FIG. 5, a plurality of minute columnar structures (micropillars) 24 are further provided at the intersection of two central portions. In this example, four micro pillars 24 are arranged in a square shape, but the number and position of the micro pillars 24 may be changed according to the purpose. The micro pillars 24 can be formed as resist micro pillars on a glass substrate by, for example, photolithography.

  In the example illustrated in FIG. 10, the flow path includes a hollow portion 51 and subsequent end portions 52 and 53, and a plurality of wall-like structures 54 are provided in the hollow portion 51 in parallel with the flow direction. The wall-like structure 54 can be similarly produced on the glass substrate by photolithography. Therefore, the electroosmotic flow flows separately by the plurality of wall-like structures 54.

  The flow cell can be downsized. FIG. 11 shows an example of a microchip. In this example, an insulator layer is formed on a glass substrate, and then portions corresponding to the flow paths for a plurality of flow cells are removed by etching by photolithography. Although not shown, an electrode is formed. Each flow path of the obtained plurality of flow cells includes a central portion 61 and two end portions 62 and 63. The flow path can also be created by etching the glass substrate by photolithography.

  Next, a specific example of a flow cell and observation of electroosmotic flow using the flow cell will be described.

  As an example, the cis-type flow cell shown in FIG. 2 was manufactured by cutting out a commercially available silicon rubber having a thickness of 1 mm as a dielectric sheet 2 with a cutter. For example, the central portion 11 of the hollow portion 3 is 23 × 10 mm, the end portions 12 and 13 are 12 × 12 mm, the width of the portion continuing from the end portion 12 to the central portion 11 is 5 mm, and the end portion from the central portion 11 is The width of the part leading to the part 13 is 7 mm. In addition, the machining accuracy may be rough. As the glass substrate 1, No. 1 of Matsunami, cover slip 52 mm × 76 mm × 0.1 mm (0.12-0.17 mm) was used. A platinum foil was used as the electrode 5. A sample of a flow marker described later was prepared and placed in the hollow portion. Then, an electric field was applied to the sample, and an image of electroosmotic flow was obtained with a SIT camera and observed in real time.

  Bead particles were used as an example of a flow marker. As the bead particles, fluorescent microbeads having a trade name of “Fluoresbrite plain YG 1.0 micron microspheres (2.6% Solids-Latex)” were purchased from Polysciences Inc, and a suspension obtained by diluting the microbeads 1000 times with pure water was used.

The measurement procedure is as follows.
1) A silicon rubber plate provided with a channel-shaped hollow portion was attached to a glass substrate (unwashed), and then the inside of the flow cell was filled with 0.8 ml of the bead suspension. It is easy to adsorb the silicon rubber plate to the glass substrate, and no liquid leakage occurs.
2) The flow cell was placed on the stage of an inverted type phase contrast fluorescence microscope, and two platinum foils (electrodes) were sandwiched between leaf springs attached to the stage, and fixed to the end portion of the flow cell. Two platinum foils were used as an anode and a cathode, respectively, and connected to a constant voltage power supply device.
3) The fluorescence (blue excitation) emitted from the bead particles in the flow cell was visualized with a color CCD camera or a black-and-white SIT camera using an oil immersion 100 × objective lens, and used as a flow marker.
4) A constant voltage of 15-30 V was applied to the platinum foil electrode to generate an electroosmotic flow on the surface of the flow cell, and the movement of the flow marker carried by the electroosmotic flow was tracked for each particle.

  The measurement results and the considerations are as follows.

  12 and 13 show the electroosmotic flow analysis using fluorescent microbeads for the conventional rectangular cell and the cis cell of FIG. 2, respectively. In the middle of the figure, the part indicated by a circle in the flow path shown in the upper part of the figure is enlarged, and the moving direction of the beads in that part is indicated by the → mark below. FIG. 14 and FIG. 15 show the change with time (relative value) of the position along the + pole direction from the + pole direction of each arbitrary bead for the two cells described above.

  In the vicinity of the anode and cathode of the flow cell, electrode reactions such as bubble formation and electrode erosion were observed when 1) the voltage was high and 2) when the conductivity of the solution was high.

  In the conventional rectangular cell, it has been assumed that the electroosmotic flow is disturbed by the electrode reaction (as a result, the conveyance and shearing by the electroosmotic flow are hindered). However, as shown in FIGS. Disturbance of electroosmotic flow was observed in the rectangular flow cell. Counter flow was identified as a cause of disturbance of electroosmotic flow. This is a phenomenon in which the electroosmotic flow turns back at the end of the flow cell (it seems to be the same mechanism that waves are reflected at the quay of the breakwater). Counter flow creates an interface between dense and sparse areas in the flow marker distribution (lower left and upper right in FIG. 12), and the direction of electroosmotic flow that should be essentially parallel to the direction of the electric field (arrow) It was random, and it was generated when it was backflowed).

  On the other hand, as shown in FIGS. 13 and 15, in the case of the U-shaped flow cell (FIG. 2), the disturbance of the electroosmotic flow observed in the conventional rectangular flow cell is eliminated, and in any region. An electroosmotic flow having a uniform flow parallel to the direction of the electric field and a flow that can be controlled by the magnitude of the voltage was realized. It was also observed that the transformer type flow cell (Fig. 3) with the two ends arranged on the opposite side across the flow cell produced the same ideal electroosmotic flow as the U-shaped flow cell. It was. The mechanism that provides such an advantage is unknown, but the flow cell is generated because the part where the counter flow occurs is away from the center of the flow cell, or the bubbles generated by the electrode reaction are trapped at the end of the U-shape. This is thought to be because it does not enter the center. From the above considerations, the end connected to the central portion through which the uniform electroosmotic flow flows may be any one that can separate the influence of the electrode reaction caused by the electrode provided at the end. Therefore, the shape and position may be appropriately designed so that the influence of the electrode reaction can be separated. The flow paths having various shapes described above are created from this viewpoint.

  Although it has been explained that a uniform flow can be realized with respect to microbeads, similarly, it has been observed that each fluorescently labeled protein (for example, RNA polymerase, antibody, etc.) molecule is transported by electroosmotic flow. Therefore, the electroosmotic flow cell can be used as a pump (conveyance device) for cells (blood) or the like. The electroosmotic flow cell is generally useful for transporting a liquid containing molecules having a size of 10 nm to 100 μm, which is handled by a gene chip or a small chemical analyzer. That is, in the electroosmotic flow cell, a liquid containing molecules having a size of 10 nm to 100 μm is introduced into the flow path, and then a constant voltage is applied to the electrodes of the electroosmotic flow cell to cause an electroosmotic flow flowing in a uniform direction. generate. Thereby, molecules having a size of 10 nm to 100 μm can be transported. Note that 10 nm is the size of one protein molecule. In addition, the flow marker particles sometimes form a huge mass, and it is visualized that the mass is conveyed by electroosmotic flow, but 100 μm is the diameter of the aggregate of such fluorescent bead particles. Equivalent to.

  In addition, in the saddle type flow cell shown in FIG. 5, it was demonstrated from the observation of the movement of the flow marker beads that the direction of the electroosmotic flow is controlled in response to turning on and off of two orthogonal electric fields.

  Chromosome was used as another example of the flow marker. Intercalator fluorescent dye YO-PRO-1 (Molecular probes) was used for DNA staining. As an example, protoplasts of fission yeast 972 h-strain were permeabilized and then adsorbed and fixed in the flow cell as described below and developed by electroosmotic flow.

  For the purpose of handling a set of DNA in one cell and preventing fragmentation due to fluid force, the artificially reinforced cytoskeleton is used as a container for DNA, using the method of Washizu et al. Occasionally, a method was used in which the container was destroyed on the substrate to remove the DNA.

  Specifically, the cell wall is first digested with an enzyme to produce a protoplast. Protoplasts are easily destroyed when subjected to osmotic shock and are difficult to manipulate, so the cross-linking agent glutaraldehyde is used to reinforce the cytoskeleton. Furthermore, DNA confined in a reinforced cytoskeleton (hereinafter referred to as cage structure) is obtained. This cage structure has an appropriate mesh size that retains long macromolecules such as DNA and allows proteins and drugs to permeate. In addition, digestion of RNA with RNase A after the cage structure is produced is effective for chromosome elongation.

  The procedure for elongation of chromosomal DNA from cage-structured cells is described. First, cells are fixed on a glass substrate by drying the cell suspension, and then the cage is used with a protease and a surfactant. Partially destroy the structure. As a result, the internal DNA is exposed, the remaining histones and the like are decomposed, and the chromosome is relaxed. Here, since a part of the DNA is fixed to the substrate via the cage structure, the DNA is pulled out of the cage structure by the electroosmotic flow and is elongated.

  When the chromosome was used as a flow marker, the chromosome was previously stained with a fluorescent dye Yo-Pro-1 (Molecular Probe) and then made into fiber, and visualized by blue excitation in the same manner as in item 3 of the measurement procedure for beads. The state of the fiber fluttering by electroosmotic flow was observed with a camera.

  FIG. 16 shows an example of elongation of chromosomal DNA of yeast (S. Pombe) using this apparatus. Since the stretched DNA does not fit in one field of view of the microscope, this picture is shown with multiple frames connected. In the photograph, a fiber having a high brightness extending in the left-right direction is DNA, and the length of extension is about 800 μm. The brightness from the center left is higher than that at both ends, which is considered to be a portion where DNA is condensed. The length of the extension obtained corresponds to three times 700 kb = about 230 μm obtained by the DNA extension method using the conventional meniscus force. Below the photo is the expanded and unfolded chromosome. In addition, since the focal point moves when the focal point of the objective lens is focused on the substrate surface and is aligned with the extended DNA, the distance of the focal point is about 2 μm. It can be seen that it is fixed to the substrate and floats at a position 2 μm away from the substrate in multiple states. Further, in the flow cell provided with a plurality of wall-like structures as shown in FIG. 10, different DNA samples can be separated and extended simultaneously by fixing the cells between the two walls.

  Therefore, the electroosmotic flow cell can be used for a DNA or linear polymer analysis method. That is, in an electroosmotic flow cell, one end of DNA or a linear polymer is fixed to a flow channel, a liquid is introduced into the flow channel, and a constant voltage is applied to the electrode of the electric flow cell to flow in a uniform direction. Generate a flow. Thereby, since a uniform shearing force is generated, DNA or linear polymer is analyzed by developing and aligning in the direction of uniform electroosmotic flow.

  On the other hand, as a comparative example, FIG. 17 shows a situation where an attempt to extend and expand yeast chromosomal DNA by electroosmotic flow generated in a conventional rectangular flow cell has failed. A chromosomal molecule group with poor development is schematically shown on the lower side of the photograph.

  As described above, the extension of the DNA from the cage structure yields an extended DNA that is fixed at one end only and is oscillated at the other end by electroosmotic flow, but this state is maintained unless voltage is continuously applied. Can not. Therefore, the extended DNA is fixed to a micro-columnar structure provided on the substrate so that the extended state can be maintained even after the voltage is turned off. That is, a plurality of pairs of electrodes and a columnar structure placed therebetween are provided on a glass substrate, and cells are placed in the vicinity thereof. When a voltage is applied to a certain pair of electrodes and the DNA extracted from the cell is stretched, a voltage is applied to another pair of electrodes to change the direction of the electroosmotic flow and adsorb the DNA to the micropillars. Or fix by winding. When this DNA treatment is used, each DNA can be separated by length.

  As an example, a micro pillar having a height of 10 μm and a diameter of 10 μm is applied to a flow path of the vertical flow cell shown in FIG. 5 using a photoresist SU-8 (MicroChem Corp), for example, in a 6 mm × 6 mm area of 100 μm. Created at intervals. Then, the cells are placed in the region where the column group exists, and the osmotic flow cell is used to create an osmotic flow in one direction (for example, right direction), thereby extending the DNA. Since the DNA extracted by the electroosmotic flow is extended at a position about 2 μm away from the substrate, the DNA is suspended in a suspension bridge shape across the two micropillars 24 without contacting the glass substrate. FIG. 18 is a photograph of the suspended yeast chromosomal DNA, and FIG. 19 schematically shows the situation. First, as shown in FIG. 20, a voltage is applied between a + and a−, and DNA having one end fixed on the substrate is extended in parallel to two micropillars 24. Next, as shown in FIG. 21, a voltage is applied in a direction orthogonal to this, and the extended DNA is suspended in the micropillar 24 and fixed. In a cell having three micropillars, the extended DNA can be wound around the three micropillars by changing the direction of the electric field. As described above, in the vertical flow cell, the orientation of the linear polymer (chromosomal DNA) having one end fixed on the substrate can be controlled as if the azimuth magnet needle is operated by a magnetic force. These results indicate that the vertical flow cell can transport the material in multiple directions (8 directions with the simplest possible combination when applying a constant voltage), and further apply shear force to the material from multiple directions. It shows that you can.

  Therefore, DNA or a linear polymer can be suspended between the columnar parts using the electroosmotic flow cell in which a plurality of columnar parts are formed in the flow path of the present invention. That is, in an electroosmotic flow cell, one end of DNA or a linear polymer is fixed to a flow channel, a liquid is introduced into the flow channel, and a constant voltage is applied to the electrode of the electroosmotic flow cell to flow in a uniform direction. Osmotic flow is generated, and DNA or linear polymer is developed and aligned in the direction of uniform electroosmotic flow. Further, by changing the voltage applied to the electrodes to change the direction of the applied electric field to change the direction of the electroosmotic flow, the developed DNA or linear polymer is suspended between the columnar parts.

  FIG. 22 shows a fully open electroosmotic flow cell device. This apparatus is similar to the U-shaped (cis-type) flow cell shown in FIGS. 1 and 2, but further has an inlet 14 for injecting a sample in the flow direction in the central portion 11, and a flow direction. A discharge port 15 for discharging the sample is provided. In a situation where a constant voltage was applied to the electrodes (not shown) at the end portions 12 and 13 and a uniform electroosmotic flow flowed in the central portion 11, the sample injected from the injection port 14 flowed uniformly in the central portion. After that, it goes out from the discharge port 15. FIG. 23 shows an example in which an injection pump (for example, a syringe) 16 is installed at the injection port 14 and a discharge pump (for example, a syringe) 17 is installed at the discharge port 15.

  There may be a plurality of inlets 14 and outlets 15, and their three-dimensional arrangement may be parallel to the substrate surface as shown in the figure, or may be vertical. Also, a combination of parallel and vertical may be used. Further, the position may be the central portion 11 or the end portions 12 and 13. Needless to say, the shape of the flow path of the flow cell apparatus is not limited to the U-shaped flow cell.

  Next, FIGS. 24 and 25 are a perspective view and a plan view of a semi-open flow cell device in which a large electroosmotic flow cell (large cell) and a small electroosmotic flow cell (small cell) are combined in a nested manner. The small cell includes a central portion 61 and two end portions 62 and 63. The end portions 62 and 63 include rectangular portions 62a and 63a and connecting portions 62b and 63b that connect the rectangular portions 62a and 63a to the side of the central portion, respectively, and the rectangular portion 62a includes an electrode (not shown). Further, the large cell includes two end portions 64 and 65 following the central portion 61, and the end portions 64 and 65 are respectively connected to the rectangular portions 64a and 65a and the connecting portions 64b and 64b that connect the side portions to the central portion. The rectangular portions 64a and 65a include electrodes (not shown). The connecting portions 64b and 65b are bent in the middle, and the electroosmotic flow linearly flows from the elongated connecting portion 64b to the elongated connecting portion 64b through the wide central portion 61. In the figure, + and-indicate the polarity of the applied DC voltage. (Note that the glass substrate is not shown in FIGS. 24 and 25, and the wall surface of the large cell is omitted in FIG. 24.) The rectangular portion 64a of the large cell is both a sample inlet and a pump. The other rectangular portion 65a of the large cell has both functions of a sample discharge port and a pump. In this flow cell apparatus, the substance conveyed by the large cell is conveyed to the small cell. Material transport is controlled by the voltage applied to the two pairs of electrodes. Needless to say, the shape of the flow path of the flow cell device is not limited to the U-shaped flow cell.

  As described above, the various flow cells of the present invention can be used for manipulation and modification of DNA and the like, but can also be combined with other functions. A small chemical analyzer (including a portable medical diagnostic device) is a chemical analysis system in which pumps, valves, sensors, and the like are miniaturized and integrated on a chip. In addition, systems that perform chemical synthesis by miniaturizing and integrating various functions such as reactors are also being studied. In such an apparatus or system, a substance can be transferred by incorporating a micro transfer apparatus using the flow cell of the present invention. Further, the flow cell capable of controlling the flow in a plurality of directions according to the present invention can be incorporated to carry out the operation of transporting the substance in multiple directions. Moreover, the substance which flows can be analyzed by arrange | positioning a sensor in the vicinity of the flow path of a flow cell.

Schematic diagram of electron osmosis flow cell device Hollow section of U-shaped (cis) flow cell Hollow section of transformer type flow cell Figure of the hollow part of a modification of the cis type flow cell Figure of hollow part of vertical flow cell Figure of the hollow part of the modification of the vertical flow cell Figure of hollow part of non-linear flow cell Figure of hollow part of multi-channel flow cell Figure of hollow part of flow cell with micro pillar Illustration of hollow part of flow cell with wall-like structure Flow cell microchip diagram Diagram showing analysis of electroosmotic flow using fluorescent microbeads for a conventional rectangular cell Diagram showing analysis of electroosmotic flow using fluorescent microbeads for cis type cell The figure which shows the time-dependent change (relative value) of the position along the + pole direction from the + pole of each arbitrary bead about the conventional rectangular cell. The figure which shows the time-dependent change (relative value) of the position along the + pole direction from the + pole of each arbitrary bead about a cis type cell. Diagram of elongation and development of yeast chromosomal DNA by electroosmotic flow generated in cis-type cells Diagram of an example of unsuccessful extension of yeast chromosomal DNA by electroosmotic flow generated in a conventional rectangular flow cell Illustration of suspended yeast chromosomal DNA Diagram showing the situation of suspended yeast chromosomal DNA Diagram showing the situation of yeast chromosomal DNA suspension Diagram showing the situation of yeast chromosomal DNA suspension Perspective view of a fully open flow cell device Perspective view of a fully open flow cell device Perspective view of semi-open flow cell device Plan view of a semi-open flow cell device

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Glass substrate, 2 Insulator sheet 2, 3 Hollow part, 4 DC constant voltage power supply device 11, 21, 21, 31, 41, 41, 51 Center part, 12, 13, 22, 23, 32, 33, 42, 43 , 52, 53 edge, 14 inlet, 15 outlet, 24 columnar structure, 54 wall structure.

Claims (21)

An electroosmotic flow cell having a flow path for electroosmotic flow,
At least the bottom surface of the flow path is made of a charged material or an artificially charged material,
The flow path is composed of a central portion having a uniform width and two end portions which are a start point and an end point of the flow path, and the central portion continues from the side portion side to the end portions in the longitudinal direction. An electroosmotic flow cell characterized by that. The electroosmotic flow cell according to claim 1, wherein the two end portions of the hollow portion are on the same side with respect to the longitudinal direction of the central portion. The electroosmotic flow cell according to claim 1, wherein the two end portions of the hollow portion are on different sides with respect to the longitudinal direction of the central portion. The electroosmotic flow cell according to claim 1, wherein a plurality of the flow paths are provided, and central portions of the plurality of flow paths intersect each other. 2. The electroosmotic flow cell according to claim 1, wherein a plurality of the flow paths are provided, and a central portion of the plurality of flow paths is provided with common end portions. An electroosmotic flow cell comprising an electroosmotic flow channel,
At least the bottom of the channel is made of a charged material or artificially charged material,
The flow path is composed of a central portion having a uniform width and two end portions serving as a start point and an end point of the flow path, and the central portion exists in a non-linear shape between the two end portions. An electroosmotic flow cell characterized by. The electroosmotic flow cell according to claim 1, wherein one or more columnar portions are provided on the bottom surface and in the flow path. Furthermore, one or more wall-shaped parts are provided on the said bottom face, and in the hollow part of the said insulator sheet, It was described in any one of Claims 1-6 characterized by the above-mentioned. Electroosmotic flow cell. It consists of a substrate of a charged material or an artificially charged material and an insulator sheet placed on the substrate, and the flow path is formed as a hollow portion of the insulator sheet. The electroosmotic flow cell according to any one of claims 1 to 8, wherein the electroosmotic flow cell is provided. The electroosmotic flow cell according to any one of claims 1 to 9, further comprising an electrode at each of the end portions. The electroosmotic flow cell according to claim 1, further comprising an inlet for injecting the sample into the channel and an outlet for discharging the sample from the channel. The electroosmotic flow cell according to claim 11, further comprising an injection pump for injecting the sample into the injection port and a discharge pump for discharging the sample from the discharge port. The electroosmotic flow cell device according to claim 10, further comprising a constant voltage power source that applies a DC constant voltage to electrodes provided at each end. An insulating sheet for forming an electroosmotic flow cell installed on the surface of a charged material or an artificially charged material, and having a central portion with a uniform width as a flow path, and a flow path An insulating sheet comprising: a hollow portion including two end portions serving as a start point and an end point of the substrate, wherein the central portion continues from the side portion side to the end portions with respect to the longitudinal direction. An insulating sheet for forming an electroosmotic flow cell installed on the surface of a charged material or an artificially charged material, and having a central portion with a uniform width as a flow path, and a flow path An insulator sheet comprising: a hollow portion including two end portions serving as a start point and an end point of the substrate, wherein the central portion exists in a non-linear shape between the two end portions. An electroosmotic flow cell chip having a plurality of flow paths provided with a bottom surface made of a charged material or an artificially charged material,
Each flow path is composed of a central portion having a uniform width and two end portions serving as a start point and an end point of the flow channel, and the central portion extends from the side to the end portions with respect to the longitudinal direction. An electro-osmotic flow cell chip, which is continued. An electroosmotic flow cell chip having a plurality of flow paths provided with a bottom surface made of a charged material or an artificially charged material,
Each flow path is composed of a central portion having a uniform width and two end portions serving as a start point and an end point of the flow path, and the central portion exists in a non-linear shape between both end portions. Electroosmotic flow cell chip. The electroosmotic flow cell chip according to claim 16 or 17, further comprising electrodes at both ends of the hollow portion. Prepare an electroosmotic flow cell with a channel formed on a charged or artificially charged material,
One end of DNA or linear polymer is fixed to the flow channel, and a liquid is introduced into the flow channel,
Analyzing by applying a constant voltage to the electrodes of an electric flow cell to generate an electroosmotic flow that flows in a uniform direction, and then deploying and aligning DNA or linear polymers in the direction of the uniform electroosmotic flow Method. Prepare an electroosmotic flow cell with a channel formed on a charged or artificially charged material,
Introducing a liquid containing molecules having a size of 10 nm to 100 μm into the flow path;
A transport method for transporting molecules having a size of 10 nm to 100 μm by applying a constant voltage to electrodes of an electroosmotic flow cell to generate an electroosmotic flow that flows in a uniform direction. An electroosmotic flow cell having a flow path formed on a charged material or an artificially charged material and having a plurality of columnar portions formed in the flow path is prepared.
One end of DNA or linear polymer is fixed to the flow channel, and a liquid is introduced into the flow channel,
Applying a constant voltage to the electrodes of the electroosmotic flow cell to generate an electroosmotic flow that flows in a uniform direction, deploying and aligning DNA or linear polymer in the direction of the uniform electroosmotic flow,
A treatment method in which the voltage applied to the electrode is changed to change the direction of the applied electric field to change the direction of the electroosmotic flow, and the developed DNA or linear polymer is suspended between the columnar parts.