JP2009162580A - Liquid drop manipulator and liquid drop manipulating method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a liquid drop manipulator of a simple structure having high design flexibility, and used for manipulating liquid drops with high accuracy without providing any physical fluid control mechanism, and a liquid drop manipulating method. <P>SOLUTION: This liquid drop manipulator is equipped with a liquid drop carrier having a carrying surface for thereon carrying liquid drops consisting of water solution containing magnetic particles, an electric field means for placing an electric field extending from below the drop carrier to above the carrying surface to fix the liquid drops on the carrying surface, a liquid drop carrier 10 having a carrying surface 10a for thereon carrying hydrophilic liquid drops 2 containing magnetic particles 2a, an electric field means 20 for placing an electric field stretching from below the drop carrier 10 to above the carrying surface 10a to fix the liquid drops 2 on the carrying surface 10a, and a magnetic field means 3 for placing a magnetic field extending from below the drop carrier 10 to above the carrying surface 10a to horizontally provide the magnetic particles 2a with force in a direction level with the carrying surface, the force being larger than the sum of fixation force of the liquid drops caused by the electric field placed thereon and surface tension of the liquid drops. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a droplet manipulation device and a droplet manipulation method.

  In chemical operations using magnetic particles, especially separation, extraction and purification processes in biochemical reaction systems, it can be used in various industrial fields by miniaturizing the devices used and controlling the reaction efficiently. Various operations in the microchemical reaction system can be performed electrically.

  A small amount of water or aqueous liquid containing magnetic particles takes the form of droplets when placed in a liquid medium such as oil that does not mix with water. For example, when magnetic silica particles having a hydrophilic surface and a particle size of about 0.1 μm to 500 μm are used, the magnetic silica particles pull the droplet in the moving magnetic field in the moving direction. This phenomenon is used for feeding a liquid reagent in a microchemical reaction system (for example, see Patent Documents 1, 2, and 3).

  When the surface of magnetic particles has a function of adsorbing specific components, etc., when separating and purifying components using this function, it is necessary to separate the magnetic particles that have adsorbed the target components from the droplets. There is. Furthermore, in order to remove impurities from the separated magnetic particles, it is necessary to pass the magnetic particles through another droplet made of a cleaning liquid.

Shikida et al. Disclose a method in which magnetic particles having a functional surface are used to separate and extract specific components on a droplet basis using magnetic force. Shikida et al. Use a physical droplet control mechanism such as a glass gate to separate magnetic particles from mother droplets in oil (see Non-Patent Document 1, for example).
International Publication No. 05/069015 Pamphlet WO03 / 058875 pamphlet JP 2006-61031 A M. Shikida, K. Takayanagi, K. Inouchi, H. Honda, and K. Sato, “For droplet manipulation of magnetic beads in microchemical analysis systems. Using wettability and interfacial tension to handle droplets of magnetic beads in a micro-chemical-analysis system ”, Sens. Actuators, B, 2006, 113, 563-569.

  However, as described above, in a device intended for chemical reaction on the basis of a minute droplet, in order to fix a droplet in a liquid medium such as oil having a specific gravity smaller than that of water, a physical barrier as in Shikida et al. It is necessary to provide a fluid control structure such as a gate. In particular, when manipulating droplets that do not contain magnetic particles, it is difficult to control movement due to a magnetic field. Therefore, a hydrophilic chemical treatment is applied to the substrate surface that physically surrounds the droplets or that contacts the droplets. It is necessary to adsorb droplets.

  However, if the fluid control structure or the substrate hydrophilic chemical treatment is performed, the apparatus becomes complicated, and the degree of freedom in designing the apparatus is lost. In addition, in the method of separating magnetic particles from the mother droplet using a glass gate or the like, the liquid particles that are not intended may be included in the separated magnetic particles depending on the size of the gate, so the droplet separation accuracy is low. Become.

  On the other hand, a droplet-based chemical reaction device containing magnetic particles may operate a plurality of droplets simultaneously. That is, one droplet may be moved and the other droplet may be fixed in place. Although it is possible to control each droplet independently using a plurality of magnets, the movement control mechanism becomes complicated. In addition, it is possible to provide a fluid control structure such as a flow path, but the structure of the apparatus becomes complicated, so the degree of freedom in design is impaired and it is difficult to realize an apparatus that can handle various chemical reactions. is there.

  In view of the above problems, the present invention provides a droplet operation device and a droplet operation method that can operate a droplet with high accuracy with a simple structure, a high degree of design freedom, without providing a physical fluid control mechanism. I will provide a.

  In order to achieve the above object, according to an aspect of the present invention, there is provided a droplet transport device having a transport surface for transporting droplets made of an aqueous solution containing magnetic particles, and first polarities different from each other disposed below the transport surface. And a second electrode, and a voltage is applied to the first and second electrodes to generate an electric field extending from below the transport surface to above the transport surface, thereby fixing the droplet on the transport surface. And magnetic field means for generating a magnetic field extending from below the droplet transfer device to above the transfer surface, and by moving the magnetic field means along the transfer surface, the magnetic particles from the droplets fixed on the transfer surface The gist of the present invention is that it is a droplet operating device for separating the liquid.

  According to another aspect of the present invention, there are provided a step of disposing a droplet made of an aqueous solution containing magnetic particles on a transport surface of a droplet transport device, and first and different polarities disposed below the droplet transport device. Applying a voltage to the electric field means including the second electrode to generate an electric field extending from the lower side of the transfer surface to the upper side of the transfer surface, thereby fixing the droplet on the transfer surface; And a step of separating magnetic particles from a droplet fixed on the transport surface by moving magnetic field means for generating a magnetic field extending from below to above the transport surface along the transport surface. This is the gist.

  According to still another aspect of the present invention, a step of disposing a droplet made of an aqueous solution containing magnetic particles on a transport surface of a droplet transport device, and a first having different polarities disposed below the droplet transport device. And applying a voltage to each of the plurality of electric field means including the second electrode to generate an electric field extending from the lower side of the transfer surface to the upper side of the transfer surface, and fixing the droplet on the transfer surface; And reciprocating the magnetic field means for generating a magnetic field extending from the lower part of the apparatus to the upper part of the conveying surface between the plurality of electric field means along the conveying surface. In contrast, the present invention is summarized as a droplet operation method that controls the moving speed of the return path to be 10 to 200 times.

  According to still another aspect of the present invention, a step of disposing a droplet made of an aqueous solution containing magnetic particles having nucleic acids bound to a surface thereof on a transport surface of a droplet transport device, and a cleaning solution on the transport surface Applying a voltage to the step and a plurality of electric field means including first and second electrodes having different polarities arranged below the droplet conveying device, an electric field extending from below the conveying surface to above the conveying surface is generated. By causing the droplets and the cleaning solution to be fixed on the conveyance surface, and by moving magnetic field means for generating a magnetic field extending from below the droplet conveyance device to above the conveyance surface along the conveyance surface. A step of separating the magnetic particles from the droplets fixed on the conveying surface, and a step of cleaning the magnetic particles by mixing the magnetic particles separated from the droplets with the cleaning solution by the movement of the magnetic field means Control And summarized in that a method.

  According to the present invention, it is possible to provide a droplet operating device and a droplet operating method that can operate a droplet with high accuracy with a simple structure, a high degree of design freedom, without providing a physical fluid control mechanism.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, the drawings are schematic, and the configuration of the apparatus and system is different from the actual one. Moreover, it is a matter of course that portions having different configurations and the like are included in the drawings. Further, the embodiments of the present invention shown below exemplify apparatuses and methods for embodying the technical idea of the present invention, and the structure, arrangement, etc. of the technical idea of the present invention will be described below. It is not something specific.

(First embodiment)
As shown in FIG. 1, the droplet operating device according to the first embodiment has a transport surface 10a (bottom surface in FIG. 1) for transporting a droplet 2 made of an aqueous solution containing magnetic particles 2a. A transport device 10, an electric field means 20 for applying an electric field extending from below the droplet transport device 10 to the top of the transport surface 10 a to fix the droplet 2 on the transport surface 10 a, and transport from the bottom of the droplet transport device 10 And a magnetic field means 3 for applying a magnetic field extending above the surface 10a to separate the magnetic particles 2a from the droplets 2 fixed on the transport surface 10a.

  The shape of the droplet transfer device 10 includes, for example, a membrane reaction vessel, a capillary, a flat plate substrate, and a flat plate substrate surface in which a space surrounded by the end portions of two films is bonded and surrounded by the film. A container having a wall, a reaction container having a lid that covers a flat substrate and a region surrounded by the wall can be used. The lid may be opened or closed in whole or in part so that a droplet containing a reagent or a sample for performing a chemical reaction can be introduced into the lid.

  In order to prevent external contamination in detection and analysis of reactants, it is particularly preferable to use a container or substrate having a completely closed reaction field as the droplet transfer device 10. As a container or a substrate having a completely closed reaction field, for example, in addition to the above-described membrane-like reaction vessel, a tubular reaction vessel having a closed channel fused at both ends, a reaction substrate, a wall, a lid, Can be suitably used.

  In the example of FIG. 1, a 10 mm high wall (side wall) is provided on a polypropylene flat plate having a thickness of 0.3 mm, a vertical width of 30 mm, and a horizontal width of 90 mm as the droplet transfer device 10 and is surrounded by the flat plate and the side wall. A rectangular parallelepiped reaction substrate (reaction vessel) whose region is sealed with a glass lid having a thickness of 5 mm is used.

  The transport surface 10a preferably has a smooth surface in contact with the droplet 2 in consideration of the ease of movement of the droplet 2, and particularly the surface roughness Ra = 0.1 μm (JIS B 0601-1994) or less. It is preferable that By setting the surface roughness Ra of the conveyance surface 10a to 0.1 μm or less, for example, when the magnetic field means 3 is brought close to the lower surface of the conveyance surface 10a and the droplet 2 is moved by the movement of the magnetic field means 3, the magnetic field means The followability of the movement of the magnetic particle 2a accompanying the movement of 3 can be improved.

  The material used for the droplet transfer device 10 is preferably a material that can be obtained at low cost from the viewpoint of being disposable or mass-produced. Further, it is preferable to use a water-repellent material in order to reduce the movement resistance when the droplet 2 moves. As such a material, for example, resin materials such as polypropylene, polytetrafluoroethylene, polyethylene, polyvinyl chloride, polystyrene, and polycarbonate can be suitably used. In order to perform optical detection in measurement of the absorbance, fluorescence, chemiluminescence, bioluminescence, refractive index change, etc. of the droplet, the droplet transport device 10 may be formed of a light-transmitting material. preferable.

  The magnetic particles 2a disposed in the droplet 2 include chemical structures that specifically bind to biologically derived polymers on the surface, such as amino groups, carboxyl groups, epoxy groups, avidin, biotin, digoxigenin, protein A, protein G, a complexed metal ion, or a magnetically responsive particle having a surface having a state of specifically binding to a polymer by electrostatic force or van der Waals force can be used.

  For example, the magnetic particle 2a is formed of a magnetic material such as magnetite, γ-iron oxide, manganese zinc ferrite, etc., and has a hydrophilic group such as a hydroxyl group, an amino group, a carboxyl group, a phosphate group, or a sulfonate group on the surface. Can be used. Specifically, magnetic particles composed of a mixture of a magnetic material and silica, magnetic particles whose surfaces are covered with silica, magnetic particles whose surfaces are covered with gold having a hydrophilic group via a mercapto group, and magnetic materials And gold particles having a hydrophilic group on the surface via a mercapto group are preferably used. The magnetic particles 2a having these functional surfaces can selectively adsorb specific components to the surface. The magnetic particle 2a preferably has an average particle size of about 0.1 μm to 500 μm.

  The magnetic particles 2 a can be taken into the droplet 2 and integrated with the droplet 2. The droplet 2 integrated with the magnetic particle 2a can move in the moving direction of the magnetic field while maintaining the droplet state by the movement of the magnetic field means 3. Here, the “droplet” in the present invention refers to a solution mass having a spherical shape or a shape close to that due to the surface tension generated by the intermolecular force of the liquid constituting the droplet.

  As the aqueous solution constituting the droplet 2, for example, water or other various aqueous solutions are used. When the droplet 2 is moved on the surface of the water-repellent substrate under a gas phase such as air, an aqueous solution containing alcohols such as ethanol can be used. In that case, the diameter is about 5 mm. It is preferable to use an aqueous solution having a surface tension sufficient to form the droplet 2. Specifically, when an ethanol aqueous solution is used, it is preferably within a concentration range of 1 (v / v) to 50 (v / v)%.

  The aqueous solution constituting the droplet 2 preferably contains a surfactant. Surfactants include nonionic surfactants and ionic surfactants. Nonionic surfactants include polyoxyethylene sorbitan monolaurate (Tween 20), octylphenol poly (ethylene glycol ether) n (Triton X-100). Nonionic surfactants are preferred in that they do not inhibit the reaction when performing a biochemical reaction in the droplet. Examples of the anionic surfactant include sodium dodecyl sulfate and N-lauryl sarcosine. By containing a surfactant, a phenomenon is observed in which the adsorption of droplets onto the conveyance surface of the droplet conveyance device becomes strong regardless of the presence or absence of magnetic particles, and fixing to a fixed position on the conveyance surface 10a is observed. This can be easily realized and the operation accuracy of the droplet 2 can be improved. The addition of the surfactant also contributes to the fixing stability of the mother droplet in the separation operation of the magnetic particle from the droplet containing the magnetic particle described later. The surfactant is preferably added to the droplet 2 in an amount of about 0.001 (v / v) to 1 (v / v)%.

  In the droplet transport device 10 of FIG. 1, a chemically inert liquid that does not mix with the droplets is enclosed. “Chemically inert liquid” refers to a substance that does not inhibit various chemical reactions such as liquid separation, mixing, dilution, stirring, heating, cooling, etc., for example, hydrocarbons such as alkanes, perfluoro Hydrophobic liquid substances such as alkanes, chemical substances in which some of the hydrogen atoms of the alkane are fluorine, mineral oils, silicone oils, fatty acids, fatty acid esters, fatty acid amides, fatty acid ketones, and fatty acid amines are listed. Among these substances, a substance having a specific gravity smaller than 1 is preferably used as the “chemically inert liquid”. By using a substance having a specific gravity smaller than 1, the droplet 2 sinks into the droplet encapsulating medium, so that the operability of the droplet 2 by the magnetic field means 3 is improved.

  For biochemical reactions conducted under relatively high temperature conditions such as reactions using thermostable enzymes, substances with low volatility, specifically mineral oils with boiling points of 200 ° C. or lower, silicone oils, fatty acid esters, By using oil or the like, volatilization of the droplet encapsulating medium and the droplet can be prevented.

  The electric field means 20 is insulated from the first and second electrodes 4a and 4b and the first and second electrodes 4a and 4b which are arranged along the lower side of the transport surface 10a and have different polarities from each other. The conductive thin plate 5 disposed along the transport surface 10a on the opposite side of the transport surface 10a with respect to the electrodes 4a and 4b.

  As the 1st electrode 4a and the 2nd electrode 4b, as shown in FIG. 1, the strip | belt-shaped electrode made from aluminum of 30 mm long, 3 mm wide, and 40 micrometers in thickness can be used, for example. The first electrode 4a and the second electrode 4b in FIG. 1 are spaced apart in parallel by an interval of 2 mm, and are connected to the power source 7, respectively. For example, when the droplet 2 is moved from the left side to the right side in FIG. 1, the first electrode 4a is connected to the negative electrode (ground), and the second electrode 4b is connected to the positive electrode. Then, by applying an electric field (electric field) between the first electrode 4 a and the second electrode 4 b from the power source 7, the droplet 2 is fixed on the transport surface 10 a above the electrode 4.

  The strength of the electric field (electric field) is preferably high enough to fix the droplet 2 on the electrode 4 when a magnetic field is applied. However, if the electric field strength is too high, insulation is insufficient and electric leakage occurs, making it difficult to fix the droplet 2. Conversely, if the electric field strength is too low, it becomes difficult to fix the droplet 2 by the electric field means 20. Therefore, the magnitude of the voltage applied between the first electrode 4a and the second electrode 4b is 0.5 to 10 kV, further 1.0 to 8 kV, and further 1.5 to 5 kV. It is preferable to apply above 10a.

  As the conductive thin plate 5, for example, a flat aluminum foil having a vertical width of 30 mm and a horizontal width of 25 mm can be used. As shown in FIG. 2C, the conductive thin plate 5 is disposed about 0.2 mm below the first electrode 4a and the second electrode 4b with the insulator 6 interposed therebetween. As the insulator 6, for example, a vinyl chloride film having a thickness of 0.2 mm can be used. By grounding the first electrode 4a and not grounding the conductive thin plate 5, it is possible to more reliably fix the droplet 2 on the electrode 4 than when the conductive thin plate 5 is grounded. It is possible to improve the operation accuracy of the droplet.

  As the magnetic field means 3, a permanent magnet or an electromagnet array can be used. The movement of the magnetic field means 3 may be controlled manually or may be electrically controlled by a control means not shown in FIG. When the magnetic field means 3 is an electromagnet array, the movement of the magnetic field means can be achieved by sequentially applying a voltage to the arrayed electromagnet.

  According to the droplet manipulation device shown in FIG. 1, the electric field generated by the electric field means 20 is transported while the magnetic field means 3 generates a magnetic field while the droplet 2 is fixed above the electrode 4a on the transport surface 10a. The magnetic particles 2a are pulled by moving the magnetic field means 3 along the surface 10a from the left side to the right side. At this time, while the aqueous solution component of the droplet 2 is fixed to the first electrode 4a that is the negative electrode, only the magnetic particles 2a and a very small amount of liquid adhering to the magnetic particles 2a are separated by the movement of the magnetic field means 3. Therefore, the magnetic particles 2a can be separated from the droplets 2 without providing a physical fluid control mechanism, and the apparatus can be simplified.

  Further, by separating the magnetic particles in the droplet 2 using the attractive force of the electric field and the magnetic field, a large amount of excess liquid can be extracted as compared with the case where the particles are physically separated by a fluid control mechanism such as a gate. Absent. Therefore, it is possible to efficiently extract the magnetic particles on which the target specific component is adsorbed.

  Furthermore, since the electric field means 20 can be freely changed in position and number according to the convenience of the operator, it can be adapted to various chemical reactions, and the degree of freedom of the apparatus is high. For example, by arranging a plurality of electric field means 20 below the transfer surface 10a, a plurality of chemical reactions can occur in one droplet transfer device 10, so that a lab-on-chip for combinatorial chemistry (lab on chip) devices.

  Further, since the magnetic field means 3 and the electric field means 20 are disposed outside the droplet transport device 10, using a completely closed reaction vessel as the droplet transport device 10 makes it a major issue to take measures against cross contamination. It can be applied to clinical tests using gene amplification.

  Next, a droplet manipulation method using the droplet manipulation device shown in FIG. 1 will be described with reference to FIGS. 2 (a) to 2 (d). The droplet manipulation method described below is an example, and it is needless to say that the droplet manipulation method can be realized by various other droplet manipulation methods.

  First, a magnetic particle suspension was prepared as a source of the droplet 2 shown in FIG. As the magnetic particle material, magnetic silica beads in a dedicated reagent kit of MFX-9600-Magnia-automatic nucleic acid extraction apparatus sold by Toyobo were used. The magnetic silica beads were suspended in 10 times the amount of pure water and then washed by centrifugation at 500 × g for 1 minute, and the washing was repeated twice. Thereafter, the obtained liquid was returned to its original volume. The particle concentration in the droplet 2 is preliminarily added to and suspended in the liquid that becomes a part of the droplet 2 so that the suspension after washing is further diluted 10 times. It was. The concentration of the magnetic particles in the magnetic particle suspension thus adjusted is 30 μg / μl.

  As shown in FIG. 2A, silicone oil (Shin-Etsu Silicone KF96-20CS) is placed in a polypropylene droplet transfer device 10 having a surface (upper surface) of a water-repellent bottom plate having a thickness of 0.8 mm as a transfer surface 10a. Was added, and 10 μl of pure water in which magnetic particles 2a were dispersed at a concentration of 30 μg / μl was sealed in silicone oil, and a droplet 2 having a diameter of 2.5 mm was placed on the transport surface 10a.

  Next, the magnetic field means 3 is brought close to the lower surface of the transport surface 10a having a flat surface and moved in the horizontal direction along the transport surface 10a. As the magnetic field means 3, for example, a neodymium permanent magnet can be used. Due to the movement of the magnetic field means 3, as shown in FIG. 2 (b), the magnetic particles 2a in the droplet 2 gather on the right side of the page, and a force that moves the entire droplet 2 to the right side acts. At this time, since the resistance between the droplet 2 and the conveyance surface 10a is small on the water-repellent conveyance surface 10a, the magnetic particle 2a does not jump out of the droplet 2, and as a result, the entire droplet 2 moves.

  The droplet 2 is further moved by the magnetic field means 3, and as shown in FIG. 2C, for example, a voltage of 8 kV is applied between the first electrode 4a and the second electrode 4b, and the first The first electrode 4a and the second electrode 4b are passed. By applying voltage, the water constituting the droplet 2 has a fixing force to the negative electrode (first electrode 4a). When the magnetic field means 3 is moved in the right direction on the paper as it is, the magnetic particles 2a are concentrated on the right side of the droplet 2 as shown in FIG. When the magnetic field means 3 is further moved to the right side, as shown in FIG. 2D, the magnetic particles 2a are separated from the droplets 2, and as a result, the droplets 2 become mother droplets 12 and small droplets 11. To be separated. The magnetic particles 2 a are collected in the small droplet 11.

  According to the droplet operation method shown in FIGS. 2 (a) to 2 (d), an electric field and a magnetic field are generated on the transport surface 10a, so that a simple structure can be used without providing a physical fluid control mechanism. The droplet 2 can be manipulated with high accuracy.

(Second Embodiment)
As shown in FIG. 3, the droplet operating device according to the second embodiment includes a first electrode 4 a having a flat plate shape (disc shape) disposed close to the transport surface 10 a of the droplet transport device 10, A first electrode 4a having a polarity different from that of the first electrode 4a, facing the first electrode 4a and having a larger outer periphery than the first electrode, a second electrode 4b having a frame shape (ring shape), the first electrode 4a and the first electrode 4a A flat plate-shaped (disc-shaped) conductive thin plate 5 that is insulated from the second electrode 4b and is disposed on the opposite side of the transport surface 10a with respect to the first electrode 4a and the second electrode 4b. Is different from the droplet operating device shown in FIG.

  As the first electrode 4a, for example, an aluminum electrode having a diameter of 5 mm and a thickness of 40 μm can be used. The first electrode 4 a is connected to the negative electrode (ground) side of the power supply 7. As the second electrode 4b, for example, an aluminum electrode having a diameter of 9 mm, a hollow portion having a diameter of 5 mm, and a thickness of 40 μm can be used. The second electrode 4 b is connected to the positive electrode side of the power source 7.

  The hollow structure of the ring-shaped second electrode 4b surrounding the first electrode 4a contributes to stabilization of the adsorption of the droplet 2 on the transport surface 10a. Although not shown, the first electrode 4a, the second electrode 4b, and the conductive thin plate 5 are insulated by a vinyl chloride film having a thickness of 0.5 mm.

  The conductive thin plate 5 is a disc-shaped aluminum foil having a thickness of 40 μm and a diameter of 9 mm, and is insulated from the first electrode 4a and the second electrode 4b through a 0.5 mm vinyl chloride film. The conductive thin plate 5 is disposed so as to overlap the second electrode 4b without being grounded.

  Various shapes such as an ellipse, a triangle, a rectangle, and a polygon can be adopted as the planar shape of the first electrode 4a. The shape of the second electrode 4b may be a frame shape having a hollow portion at the center, and may be, for example, an ellipse, a triangle, a rectangle, or a polygon. The rest of the configuration is substantially the same as that of the droplet manipulation device shown in FIG.

  According to the droplet manipulation device using the electric field means 20 shown in FIG. 3, a magnetic field is generated while an electric field is generated and the droplet 2 is fixed on the transport surface 10a above the flat plate-like first electrode 4a. By pulling the magnetic particles 2a horizontally on the transport surface 10, the droplets can be manipulated with high accuracy with a simple structure without providing a physical fluid control mechanism.

  Further, as shown in FIG. 3, by using the flat or frame-shaped first electrode 4a and second electrode 4b, the band-shaped first electrodes 4a and 4b as shown in FIG. 1 are used. Since the movement of the droplet 2 in the extending (banding) direction of the electrode that can occur can be suppressed, the control efficiency of the droplet 2 can be further improved.

  Next, a droplet operation method using the electric field means 20 shown in FIG. 3 will be described with reference to FIGS. 4 (a) to 4 (d). The droplet operation method described below is an example, and it is needless to say that it can be realized by various other droplet operation methods.

  First, silicone oil (Shin-Etsu Silicone KF96-20CS) is put into a polypropylene droplet transfer device 10 having a water-repellent bottom plate (thickness 0.8 mm) as a transfer surface 10a. An electric field (electric field) is generated on the transport surface 10a via the first electrode 4a and the second electrode 4b.

  After application of the electric field, a magnetic particle suspension containing 0.01 (v / v)% surfactant (Tween 20) and having a magnetic particle concentration of 30 μg / μl is transferred by a micropipette to the transport surface 10a immediately above the first electrode 4a. The droplet 2 shown in FIG. 4A was formed in the silicone oil. A cleaning droplet 8 for cleaning magnetic particles extracted from the droplet 2 was also disposed on the transport surface 10a.

  With the electric field generated, the magnetic field means 3 (not shown) is approached from below the transport surface 10a, and the magnetic field means 3 is moved along the transport surface 10a from the left side to the right side of the paper at a speed of 2 mm per second. Due to the movement of the magnetic field means 3, as shown in FIG. 4B, the magnetic particles 2 a in the droplet 2 gather on the right side, and a force that moves the entire droplet 2 to the right side acts.

  With the electric field generated, the magnetic field means 3 is used to move the liquid droplet 2 further to the right in the drawing, and the liquid droplet 2 is changed into a small liquid droplet 11 and a mother liquid droplet 12 as shown in FIG. Separate. The small droplet 11 was accompanied by magnetic particles 2a and some solvent (water) of the droplet 2. Further, the magnetic field means 3 is moved rightward on the paper surface, and as shown in FIG. 4D, the small droplet 11 and the cleaning droplet 8 are combined to form the mixed droplet 18 including the magnetic particles 2a. did.

  According to the droplet operation method shown in FIGS. 4A to 4D, an electric field and a magnetic field are generated on the transport surface 10a to operate the movement of the droplet 2, thereby providing a physical fluid control mechanism. Without providing, magnetic particles in the droplet can be separated with high accuracy with a simple structure. The operations shown in FIGS. 4 (a) to 4 (d) are not limited to the above-described separation of the droplets, but may include various operations such as droplet separation, dilution, or extraction of specific components adsorbed on the magnetic particle surface. Of course, it can be used for the chemical reaction.

(Third embodiment)
As shown in FIGS. 5A to 5E, a plurality of droplet manipulation devices according to the third embodiment are arranged apart from each other along the conveyance surface 10a of the droplet conveyance device 10. The point which has the electric field means 20 made is different from the droplet operating device according to the first and second embodiments.

  On the lower surface side of the transport surface 10a, a first electrode 4a, a second electrode 4b having a polarity different from that of the first electrode 4a, facing and spaced apart from the first electrode 4a, and the first electrode 4a And the electric field means 20 including the conductive thin plate 5 that is insulated from the second electrode 4b and faces the transport surface 10a is disposed. An insulator 6 is formed around the first electrode 4a and the second electrode 4b. The conductive thin plate 5 that is insulated from the first electrode 4a and the second electrode 4b via the insulator 6 is formed of a single aluminum foil or the like, and is arranged in parallel along the transport surface 10a. It arrange | positions so that the electrode 4 may be covered, respectively.

  For example, as shown in FIG. 5A, each of the three first electrodes 4a arranged side by side along the transport surface 10a is arranged so as to be separated from the center of the electrode by about 13 mm. . Others are substantially the same as those of the droplet manipulating apparatus according to the first and second embodiments, and thus redundant description is omitted.

  According to the droplet operation device shown in FIGS. 5A to 5E, a plurality of electric field means 20 are arranged side by side along the transfer surface 10a, thereby causing a plurality of chemical reactions to proceed on one transfer surface 10a. Therefore, a highly flexible system can be constructed in the design of the chemical reaction circuit.

  Next, a droplet manipulation method using the droplet manipulation device shown in FIGS. 5 (a) to 5 (e) will be described. The droplet manipulation method described below is an example, and it is needless to say that the droplet manipulation method can be realized by various other droplet manipulation methods.

  First, silicone oil (Shin-Etsu Silicone KF96-20CS) was placed in a polypropylene droplet transfer device 10 having a water-repellent bottom plate (thickness 0.8 mm) as a transfer surface 10a. Next, as shown in FIG. 5A, droplets 2 and cleaning droplets 8 and 9 were respectively dropped on the transport surface 10a immediately above the three first electrodes 4a. On the left first electrode 4a in the figure, for example, the concentration of xylene cyanol 0.05% (w / v), 0.01% (v / v) Tween 20 aqueous solution and magnetic particles 2a is 30 μg / μl. Droplet 2 made of the mixed solution was dropped. Droplets (cleaning droplets) 8 and 9 made of 0.01% (v / v) Tween 20 aqueous solution were dropped on the center-side and right-side first electrodes 4a in FIG. The amount of droplets 2, 8, and 9 dropped was 10 μl. The procedure for dropping the droplets 2, 8, and 9 was the same as in the second embodiment. The applied voltage to the first electrode 4a and the second electrode 4b was all 8 kV.

  In a state where an electric field (electric field) is applied to the conveying surface 10a by the electric field means 20, the magnetic field means 3 is brought close to the lower surface of the conveying surface 10a, and as shown in FIG. 5B, the paper surface along the conveying surface 10a. By moving the magnetic field means 3 from the left direction to the right direction at a speed of about 2 mm / s, the magnetic field means 3 generates a force larger than the fixing force of the droplet 2 on the first electrode 4a and the surface tension of the droplet 2 by applying an electric field. Was applied to the magnetic particle 2a in the horizontal direction of the transport surface, and the droplet 2 containing the magnetic particle 2a was separated into the mother droplet 12 and the small droplet 11 containing the magnetic particle 2a. Further, the magnetic field means 3 is moved rightward on the paper surface to combine the small droplet 11 and the cleaning droplet 8 to form a mixed droplet 18 including magnetic particles 2a as shown in FIG. 5C.

  In a state where an electric field is applied to the conveying surface 10a by the electric field means 20, respectively, the magnetic field means 3 is moved at a speed of about 2 mm per second along the conveying surface 10a in the right direction on the paper as shown in FIG. The mixed droplet 18 containing the particles 2a was separated into the mother droplet 14 and the small droplets 13 containing the magnetic particles 2a. Further, the small liquid droplet 13 is moved rightward on the paper surface by the magnetic field means 3 so as to be combined with the cleaning liquid droplet 9 to form a mixed liquid droplet 19 as shown in FIG. 5E, thereby cleaning the magnetic particle 2a.

  According to the droplet operation method shown in FIGS. 5A to 5E, the magnetic particles 2a in the droplet 2 can be accurately detected with one transport surface 10a without providing a physical fluid control mechanism. Can be separated and washed.

-Liquid transport method using droplet transport device-
By changing the moving speed of the magnetic field means 3, it is possible to adjust the volume of the liquid following the magnetic particles 2a when separating the magnetic particles from the mother droplet. Using this method, it is possible to transport liquid between a plurality of droplets.

  6 (a) to 6 (d) show a case where magnetic particles (iron powder, particle size 45 μm) are added to the droplet 30 fixed on the conveying surface by an electric field means (not shown), and magnetic field means is applied from the lower side of the conveying surface. 3 shows a case where the magnetic particles (aggregate of magnetic particles) 31 in the droplet 30 are pulled by moving the 3 from the right side to the left side in the drawing, and the small droplets are separated from the droplet 30. 6A and 6B show an example in which the magnetic field means 3 is moved along the transport surface at 1 mm per second, and FIGS. 6C and 6D show the magnetic field means 3. An example of moving along the conveying surface at 200 mm per second is shown.

  As shown in FIGS. 6B and 6D, the fraction of the liquid adhering to the magnetic particles 31 increases as the moving speed of the magnetic field means 3 is made slower. As shown in FIG. 6 (c), when the droplet 30 containing the magnetic particles 31 is moved at a high speed, the shape of the droplet becomes streamlined and the tail liquid cannot follow the magnetic particles 2a. It is considered that the amount of liquid droplets attached to the periphery of the particles 31 is reduced. Further, it is considered that when the magnetic field means 3 is moved at a high speed, the magnetic force line direction of the magnetic particles 2a is inclined from the vertical direction to the oblique direction, so that the liquid holding force of the magnetic particles 2a is reduced.

  FIGS. 7A to 7G show examples in which the phenomenon shown in FIGS. 6A to 6D is used for feeding a liquid between the plurality of electric field means 200a and 200b. 7 (a) to 7 (g), the electric field means 200a including the first electrode 44a and the second electrode 44b insulated from each other by the insulator 66a below the transport surface, and the insulator 66b. An electric field means 200b including a first electrode 45a and a second electrode 45b that are insulated from each other is disposed. FIG. 7 illustrates the middle of the operation procedure, and the droplet 40 does not exist at the start of the operation.

  As shown in FIG. 7A, in the state where the liquid droplets 30 and 40 are fixed on the transport surface by applying a voltage to the first electrodes 44a and 45a and the second electrodes 44b and 46b, the magnetic field means 3 Are slowly moved from the lower side of the droplet 30 along the conveying surface from the left side to the right side. The method for fixing droplets by the electric field means 200a and 200b is as described in the first to third embodiments.

  As a result, as shown in FIG. 7B, the magnetic particles 31 are extracted from the droplets 30 with a part of the droplets 30 attached around the magnetic particles 31. By moving the magnetic field means 3 as it is from the left side of the drawing to the right, the magnetic particles 31 accompanied by a part of the droplet 30 are combined with the droplet 40 as shown in FIG.

  As shown in FIG. 7 (d), this time, in a state where the droplet 40 is fixed on the transport surface, the magnetic field means is moved from the lower side of the transport surface at a speed higher than the speed at which the magnetic means is moved in FIG. 7a is moved in the direction opposite to the moving direction of FIG. 7A, that is, moved from the right side to the left side. By moving the magnetic field means at a speed faster than the moving speed in FIG. 7A, as shown in FIG. 7E, a smaller amount of liquid than that shown in FIG. The magnetic particles 31 are separated from the droplets 40 while being attached to the surroundings. Thereafter, the magnetic field means is moved from the right side to the left side as it is, and the magnetic particle aggregate 31 is united with the droplet 30 as shown in FIG.

  By repeating the operations shown in FIGS. 7A to 7F many times, the liquid in the droplet 30 is gradually taken into the droplet 40 as shown in FIG. As a result, liquid feeding from the electric field means 200a to 200b can be realized. Note that the total amount of liquid transported by the magnetic particles 31 increases as the number of times the magnetic particles 31 are reciprocated between the droplets 30 and 40 by repeating the operations shown in FIGS. To do.

  In microfluidics using microdroplets, it is known that liquid or droplets can be fed by attaching water or an aqueous solution to magnetic particles with a hydrophilic surface and manipulating the magnetic particles with a magnetic force. ing. At that time, the amount of liquid surrounding the magnetic particles in the small droplets increases according to the amount of magnetic particles, so if you want to send an arbitrary volume of liquid, control by adjusting the amount of magnetic particles Yes. The control range of the liquid feeding amount is about 0.01 to 4 μl although it depends on the type of magnetic particles.

  However, when liquid is transferred from a droplet at one point to a droplet at another point, the amount of liquid transferred is limited by the separation of one small droplet and the combination of droplets. It is necessary to add and separate the mother droplet many times and combine it with the target droplet. By repeating this work over and over, magnetic particles accumulate in the target droplets, making it difficult to remove the magnetic particles from the droplets that have accumulated magnetic particles. There is a case.

  On the other hand, according to the liquid droplet feeding method according to the embodiment of the present invention, as shown in FIGS. 7A to 7G, magnetic particles are transferred from one electric field means to the other electric field means. The moving speed of the magnetic field means 3 when moving the magnetic field and the moving speed of the magnetic field means 3 when moving the magnetic particles from the other electric field means to the one electric field means are controlled to different speeds. That is, the moving speed of the magnetic field means 3 is opposite to the moving speed of the forward path (movement from the electric field means for discharging the liquid to the electric field means for storing the liquid), whereas the moving speed of the magnetic field means 3 is the electric field means for storing the liquid. By increasing the movement speed of the movement of the liquid to the electric field means for discharging the liquid, the amount of droplets adhering around the magnetic particles is biased. An arbitrary amount of liquid can be fed by reciprocating the magnetic particles between a plurality of droplets in a state where the amount of droplets is biased.

  In this embodiment, it is preferable that the moving speed of the magnetic field means is 10 to 200 times the moving speed of the return path relative to the moving speed of the forward path. More preferably, the moving speed of the return path is 50 to 100 times the moving speed of the forward path. Specifically, the forward path is preferably 0.1 to 10 mm / sec, more preferably 0.5 to 5 mm / sec, and the return path is preferably 50 to 300 mm / sec. More preferably, the forward path is 1 to 3 mm / sec and the backward path is 150 to 200 mm / sec.

  FIG. 8 shows an example of a droplet operating device using the liquid feeding method shown in FIGS. On the transport surface 10a of the rectangular parallelepiped droplet transport apparatus 10, a film 21 having a thickness of 0.05 mm is disposed. As the film 21, for example, a Teflon (registered trademark) film can be used.

  Below the transport surface 10a, two first electrodes 4a each having a disc (flat plate) shape are disposed on the transport surface 10a so as to be spaced apart from each other in the horizontal direction. A second electrode 4b having a ring (frame) shape having a polarity different from that of the first electrode 4a is disposed under the first electrode 4a. A disk (flat plate) -like conductive thin plate 5 insulated from the first electrode 4a and the second electrode 4b is disposed under the second electrode 4b. The electrode of the first electrode 4 a is connected to the negative electrode (ground) side of the power source 7, and the second electrode is connected to the positive electrode side of the power source 7.

  Although not shown in FIG. 8, as shown in FIG. 9, the periphery of the first electrode 4a and the second electrode 4b is covered with an insulator 6 such as vinyl chloride. The conductive thin plate 5 is formed of a single aluminum foil or the like having a thickness of 45 μm. In the example of FIG. 9, the diameter l4 of the conductive thin plate 5 is 9.5 mm, the outer diameter l2 of the second electrode 4b is 9.0 mm, the ring width l3 of the second electrode 4b is 1.2 mm, The diameter l1 of one electrode 4a is 5.5 mm. The thickness t1 of the film 21 is 0.05 mm, the thickness t2 of the bottom surface of the droplet operating device 10 is 0.3 mm, and the first electrode 4a and the second electrode in the thickness direction of the bottom surface of the droplet operating device 10 are used. The inter-electrode distance t3 was 0.36 mm, and the distance t4 between the second electrode 4b and the conductive thin plate 5 in the thickness direction of the bottom surface of the droplet operating device 10 was 0.18 mm. Although not shown in FIGS. 8 and 9, when liquid is fed using the droplet operating device 10 shown in FIGS. 8 and 9, it is along the transport surface 10 a from below the conductive thin film 5. The magnetic field means 3 such as a magnet is moved.

  The graph of FIG. 10 shows that the magnetic field means 3 is controlled between the two first electrodes 4a using the droplet operating device 10 shown in FIGS. 8 and 9, and magnetic particles (iron powder having a particle diameter of 45 μm) are magnetic field means. 3 shows the amount of liquid fed per reciprocating movement when reciprocating at a moving speed of 3. FIG. The moving speed of the magnetic field means 3 was measured with the forward path being 1 mm per second and the return path being 50 mm per second, 100 mm per second, and 200 mm per second, respectively. The horizontal axis of the graph in FIG. 10 indicates the amount of magnetic particles, and the vertical axis of the graph indicates the liquid volume carried by the magnetic particles per reciprocating movement. In the experiment, a droplet made of 50 μl of pure water in silicon oil (Shin-Etsu Silicone KF96-20CS) was placed on one of the first electrodes 4a, and a DC voltage (8 kV) was applied to each of the two first electrodes 4a. ) Is applied to fix the droplet on the transport surface, and the magnet is operated horizontally from below the transport surface to the transport surface to control the movement of the magnetic particles on the transport surface. The magnetic particles were reciprocated 10 times between two droplets, and the average liquid feeding amount per time was calculated and plotted on a graph. As shown in the results of FIG. 10, it can be seen that the faster the return path is, the larger the amount of magnetic particles is, and the more the amount of liquid fed increases.

  The graph of FIG. 11 is a graph in which the amount of liquid feeding is measured using two types of magnetic particles having different particle sizes, with the traveling speed of the outgoing path being 1 mm per second, the moving speed of the returning path being 50 mm per second and 200 mm per second. The experimental conditions were the same as those in FIG. 10 except for the difference in the particle size of the magnetic particles. As magnetic particles, iron powder having a particle size of 45 μm and a particle size of 150 μm was used. As shown in FIG. 11, the liquid feeding amount tends to increase as the particle size of the magnetic particles increases. Further, in the case of feeding a liquid of several tens of nL to 100 nL, it can be achieved by changing the amount of magnetic particles from 50 μg / 50 μL to 100 μg / 50 μL. In the case of a biochemical reaction typified by a gene amplification reaction, transport of nL order liquid is very effective.

  According to the liquid transport method according to the embodiment of the present invention, in the droplet operating device 10 shown in FIG. 8 and FIG. 9, by controlling the moving speed, the number of movements, the particle diameter, and the particle amount of the magnetic particles, A certain amount of liquid can be fed.

-Nucleic acid isolation method-
The droplet manipulation method according to the third embodiment described above is applied to a nucleic acid isolation method (JP-A-2-289596) using silica particles and chaotropic salt, and nucleic acid amplification is performed using the isolated gene as a template nucleic acid. An example in which the polymerase chain reaction (PCR), which is a typical example of the reaction, is described below. In the nucleic acid isolation method according to the embodiment of the present invention, a case where the droplet manipulation device shown in FIGS. 5A to 5E is used will be described as an example. An oral swab was prepared as a biological sample containing nucleic acid.

  The configuration of each droplet in FIG. 5A is as follows. The droplet 2 on the left side of the paper was a droplet composed of a biological sample and a cell lysate, and was a mixture of 2 μl of 6M guanidine thiocyanate aqueous solution and 2 μl of oral swab. As the magnetic particles 2a in the droplet 2, magnetic silica particles were added to a concentration of 1 μg / μl. A 200 mM potassium chloride and 50 mM Tris-hydrochloric acid pH 8.0 aqueous solution (50 μl) was prepared using the droplet 8 at the center of the paper surface as a magnetic silica particle washing solution. The droplet 9 on the right side of the paper was 3 μl of the PCR reaction solution. The composition of the PCR reaction solution will be described later. The moving speed of the magnetic field means 3 was 2 mm / second.

  First, as shown in FIG. 5A, the oral mucosa cells contained in the oral swab were dissolved in the droplet 2, and the nucleic acid released in the mixed droplet was adsorbed on the surface of the magnetic particle 2a. By moving the magnetic field means 3 along the transport surface 10a, as shown in FIG. 5B, the magnetic droplet 2a having the nucleic acid adsorbed on the surface and the small droplet 11 with a small amount of guanidine thiocyanate aqueous solution are converted into the mother droplet 12. Separated from. Since guanidine thiocyanate inhibits a gene amplification reaction connected to this step, which will be described later, by further moving the magnetic field means 3 in the right direction on the paper surface, the cleaning droplet 8 made of the cleaning liquid is passed, and FIG. As shown, mixed droplets 18 were formed.

  The magnetic field means 3 was further moved rightward on the paper surface, and the small droplets 13 were separated from the mixed droplets 18 as shown in FIG. The magnetic particles 2a were accompanied by a small amount of washing liquid while adsorbing nucleic acids on the surface. The separated small droplet 13 is accompanied by potassium chloride and Tris-hydrochloric acid buffer (pH 8.0), but these components do not greatly inhibit the enzyme reaction. By further moving the magnetic field means 3 to the right in the drawing, the separated small droplet 13 is mixed with the droplet 9 made of the PCR reaction solution as shown in FIG. Formed.

The PCR reaction solution constituting the droplet 9 includes 20 mM Tris-HCl (pH 9.5), 8 mM MgCl ₂ , 200 μM each of dATP, dCTP, dGTP and dTTP, 0.2 (wt / v)% BSA, 0.4 each. A PCR reaction solution containing μM primer and 2.5 units / 50 μl Taq DNA polymerase (TaKaRa Taq: Takara shuzo, Kyoto, Japan) was prepared. The template gene was β-actin gene, and as primers, a first β-actin detection primer consisting of the base sequence of SEQ ID NO: 1 and a second β-actin detection primer consisting of the base sequence of SEQ ID NO: 2 were prepared.

  The mixed droplet 19 is reciprocated by a magnet along a conveying surface having a linear temperature gradient from 94 ° C. to 60 ° C., and the temperature of the droplet is 94 ° C. 2 seconds, 60 ° C. 1 second, and 72 ° C. 5 seconds. PCR was performed while controlling the position of the droplets so that the temperature cycle consisting of 40 cycles was repeated. After completion of the PCR, amplification products (151 bases) from the β-actin gene could be confirmed by performing agarose gel electrophoresis on the reaction products (see FIG. 12). Here, in FIG. 12, the left lane is a molecular weight marker, and the right lane is an agarose gel electrophoresis image of the reaction product.

  According to the nucleic acid isolation method according to the embodiment of the present invention, the sample extraction with the magnetic particles 2a, the nucleic acid extraction purification and the gene amplification reaction could be performed in the same container.

  In the method for isolating nucleic acids according to the embodiments of the present invention, biological samples for isolating nucleic acids include animal and plant tissues, body fluids, excreta, etc., and the body fluids are blood-derived samples such as whole blood, plasma, and serum. , Including saliva. As the cell lysate, any one or a combination of guanidine salt, potassium iodide, sodium iodide, sodium (iso) thiocyanate, and urea can be used.

  As mentioned above, although this invention was described by embodiment of this invention, it should not be understood that the statement and drawing which make a part of this indication limit this invention. From this disclosure, various alternative embodiments, examples, and operational techniques will be apparent to those skilled in the art. The technical scope of the present invention is determined only by the invention specifying matters according to the appropriate claims from the above description, and can be modified and embodied without departing from the scope of the invention in the implementation stage.

(Explanation of sequence listing)
Sequence number 1 and 2 described in the sequence table of this specification show the following sequences.

[SEQ ID NO: 1] Base sequence of first β-actin detection primer.

[SEQ ID NO: 2] Base sequence of second β-actin detection primer.

It is a perspective view which shows an example of the droplet operation apparatus which concerns on the 1st Embodiment of this invention. FIG. 2A to FIG. 2D are schematic views showing an example of a droplet manipulation method according to the first embodiment of the present invention. It is a perspective view which shows an example of the droplet operation apparatus which concerns on the 2nd Embodiment of this invention. FIG. 4A to FIG. 4D are schematic views showing an example of a droplet operating method according to the second embodiment of the present invention. FIG. 5A to FIG. 5E are schematic views showing an example of a droplet manipulation device according to the third embodiment of the present invention. FIG. 6A to FIG. 6D are schematic views showing the principle of the droplet transport method according to the embodiment of the present invention. FIG. 7A to FIG. 7G are schematic views showing an example of a method for feeding a predetermined amount of liquid by reciprocating magnetic particles between two droplets fixed on the transport surface. It is the schematic which shows an example of the droplet operation apparatus suitable for the conveyance method of the droplet which concerns on embodiment of this invention. It is sectional drawing seen from the AA direction of FIG. FIG. 8 and FIG. 9 show the relationship between the amount of liquid transported per reciprocation and the amount of magnetic particles when magnetic particles are reciprocated between two droplets fixed on an electrode using the droplet operation shown in FIG. It is a graph. The total liquid amount and magnetic particle amount when the magnetic particles are reciprocated between two droplets fixed on the electrode using the droplet operation shown in FIGS. 8 and 9 and the speed and particle diameter of the return path are changed. It is a graph which shows the relationship. It is an agarose gel electrophoresis image about the reaction product which PCR is performed about the droplet obtained by the isolation method of the nucleic acid using the droplet operating device concerning a 3rd embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 2 ... Droplet 2a ... Magnetic particle 3 ... Magnetic field means 4a ... 1st electrode 4b ... 2nd electrode 5 ... Conductive thin plate 6 ... Insulator 7 ... Power supply 8 ... Droplet 9 ... Droplet 10 ... Droplet conveyance apparatus DESCRIPTION OF SYMBOLS 10a ... Conveyance surface 18 ... Mixed droplet 19 ... Mixed droplet 20 ... Electric field means

Claims (12)

A droplet transport device having a transport surface for transporting droplets made of an aqueous solution containing magnetic particles;
A first electrode and a second electrode having different polarities disposed below the transport surface, and applying a voltage to the first and second electrodes, from below the transport surface to above the transport surface; An electric field means for fixing the droplet on the transport surface by generating an electric field extending;
Magnetic field means for generating a magnetic field extending from below the droplet transfer device to above the transfer surface,
A droplet operating apparatus, wherein the magnetic particles are separated from the droplet fixed on the transport surface by moving the magnetic field means along the transport surface.   2. The droplet operating device according to claim 1, wherein the electric field means applies a voltage of 0.5 to 10 kV between the first electrode and the second electrode.   3. The droplet operating device according to claim 1, wherein the first and second electrodes are band-like electrodes arranged in parallel with each other below the transport surface. The first electrode and the second electrode are flat electrodes,
3. The second electrode according to claim 1, wherein the second electrode is disposed further below the first electrode below the transport surface and has a larger surface area than the first electrode. Droplet operation device. The first electrode is a flat electrode;
The second electrode is a frame-shaped electrode, and is disposed below the first surface and below the transport surface, and is larger than the first electrode. The droplet operation device according to claim 1 or 2.   A conductive thin plate is further provided below the transport surface, facing the transport surface across the first and second electrodes and insulated from the first and second electrodes. Item 6. The droplet manipulation device according to any one of Items 1 to 5.   The droplet operating device according to claim 1, wherein the first electrode is grounded, and the second electrode is a positive electrode. A plurality of the electric field means are disposed along the conveying surface;
The magnetic field means moves between the electric field means at a moving speed of the return path of 10 to 200 times with respect to the moving speed of the forward path. Droplet manipulation device. Arranging a droplet made of an aqueous solution containing magnetic particles on a transport surface of the droplet transport device;
A voltage is applied to an electric field means including first and second electrodes having different polarities arranged below the droplet transfer device to generate an electric field extending from below the transfer surface to above the transfer surface. Fixing the droplet on the transport surface,
The magnetic particles are separated from the droplets fixed on the transport surface by moving magnetic field means for generating a magnetic field extending from below the droplet transport device to above the transport surface along the transport surface. And a step of performing a droplet operation. Arranging a droplet made of an aqueous solution containing magnetic particles on a transport surface of the droplet transport device;
A voltage is applied to each of a plurality of electric field means including first and second electrodes having different polarities arranged below the droplet transfer device to generate an electric field extending from below the transfer surface to above the transfer surface. Fixing the droplets on the transport surface;
Reciprocating along the transport surface between the plurality of electric field means, magnetic field means for generating a magnetic field extending from below the droplet transport device to above the transport surface,
A droplet operating method, wherein the moving speed of the magnetic field means is controlled so that the moving speed of the return path is 10 to 200 times the moving speed of the forward path. Disposing droplets made of an aqueous solution containing magnetic particles having nucleic acids bound to the surface on the transport surface of the droplet transport device; and
Placing a cleaning solution on the transport surface;
A voltage is applied to a plurality of electric field means including first and second electrodes having different polarities arranged below the droplet transfer device, and an electric field extending from below the transfer surface to above the transfer surface is generated. Fixing the droplets and the cleaning solution on the transport surface by generating, respectively,
The magnetic particles are separated from the droplets fixed on the transport surface by moving magnetic field means for generating a magnetic field extending from below the droplet transport device to above the transport surface along the transport surface. And steps to
A step of mixing the magnetic particles separated from the droplets with the cleaning solution by the movement of the magnetic field means, and cleaning the magnetic particles.   A step of preparing the magnetic particles having nucleic acid bound to the surface by adding magnetic particles to a droplet composed of a biological sample and a cell lysate and binding the nucleic acid contained in the biological sample to the surface of the magnetic particle. The droplet operation method according to claim 11, further comprising: