JP4997522B2 - Method for producing single diameter alginate microbeads and apparatus for producing the same - Google Patents

Description

The present invention relates to a manufacturing method and manufacturing equipment of a single diameter alginate microbeads used in the fields such as micro-chemical engineering, drug delivery, cell culture and regeneration medical engineering (MAB).

Alginic acid polymers have good biocompatibility and are versatile materials such as food and cell encapsulation. There are many examples of using this for drug delivery and regenerative medical engineering materials.
By the way, encapsulating a cell in a hydrogel provides protection to the cell and is related to bioreactor and tissue engineering (see Non-Patent Document 1 below). Alginic acid is the most commonly used and studied hydrogel because of its biocompatibility, availability and relatively low cost. If cells can be encapsulated in alginic acid to form an array, many cells can be visualized and analyzed simultaneously while encapsulating the cells in a protective film. There will be many applications in the study of cellomics.

  The production of fine single-diameter (monodisperse) capsules is one that has been well studied (see Non-Patent Document 2 below). When cells are encapsulated in microbeads, the exchange of nutrients to the capsules and the discharge of unwanted materials can be accomplished in a short time by diffusion, improving the viability of the encapsulated cells (see below). Non-Patent Document 3). Furthermore, by monodispersing the microbeads, the number of encapsulated cells in each microbead can be controlled more accurately.

The formation of small non-uniform diameter (polydisperse) alginate microbeads to encapsulate cells using sodium alginate and calcium chloride droplets in a microdevice is described by Sugiura et al. (See Non-Patent Document 4 below). ).
WO 02/068104 A1 M.M. M.M. Stevens et al. , "A Rapid-Curning Alginate Gel System: Utility in Perioseum-Derived Cartridge Tissue Engineering", Biomaterials, 25, pp. 887-894, 2004. G. Olive et al. "Cell Encapsulation: Promise and Progress" Nature Medicine, 9, pp. 104-107, 2003. W. M.M. Ku (u is an umlaut) htriber, R .; P. Lanza, and W.L. L. Chick, Cell encapsulation technology and therapeutics, Birkha (a with umlaut) user Boston, 1999. S. Sugiura, T .; Oda, Y .; Izumida, Y. et al. Aoyagi, M .; Satake, A.M. Ochiai, N .; Ohchichi, M .; Nakajima, “Size Control of Calcium Alginate Beads Containing Living Cells using Micro-Nozzle Array”, Biomaterials, 26, pp. 228 3327-3331, 2005. D. Poncelet, “Production of Alginate Beads by Emulsification / Internal Gelation”, 2001, Anals of the New York Academy of Sciences, 944. 74-82. A Tixier-Mita, Y. et al. Mita, K.M. Cozic, F.M. M.M. , B. LePioufle, and H.M. Fujita, “To place cells an array using aspirate technique,” Micro Total Analysis Systems, Nara, Japan, 2002. B. Le. Piofull, P.M. Surbled, H.M. Nagai, Y .; Murakami, H .; S. Chun, E .; Tamiya, and H.M. Fujita, “Living cells captured on a biosystem developed to DNA injection,” Materials Science and Engineering: C, vol. 12, pp. 77-81, 2000. B. M.M. Taff and J.M. Voldman, "A Scalable Row / Column- Addressable Dielectricphoretic Cell-Tapping Array", Micro Total Analysis Systems, Boston, USA, pp. 865-867, 2005. F. M.M. White, Fluid Mechanics, 3rd ed. , New York, NY: McGraw Hill, 1994. Stefan Fielder et al. , “Dielectricphoretic Sorting of Particles and Cells in a Microsystem”, Anal. Chem. 1998, 70, pp. 1909-1915

However, in order to improve the uniformity and size distribution of microbeads, it is necessary to solve the following two problems. That is, (1) the problem of deformation when polyvalent cations are externally added to the fine sodium alginate droplets for gelation, and (2) the problem of unnecessary fusion of the sodium alginate droplets before gelation .
Therefore, in the present invention, alginic acid is repeatedly studied with reference to the previously proposed internal gelation method (see Non-Patent Document 5) and the T-shaped intersection droplet generation method (see Patent Document 1). Microbeads (MAB) were generated.

  It also attempts to encapsulate cells in MAB for cell assay. Fluidic methods such as the suction method (see Non-Patent Document 6), biochemical techniques such as chemical patterning on the gold surface (see Non-Patent Document 7), dielectrophoretic traps (capturing) (Non-Patent Document 6) A cell array based on Patent Document 8) has been reported so far. However, many of these cell arrays require complex creation procedures and controls.

  Therefore, the present inventors propose a passive microfluidic trap device (a microbead capturing device comprising microchannels) in order to achieve the fixation of “one bead per trap”. Research is being conducted towards the development of microbead-based cell assay systems that are gentle and easy to handle, as well as techniques for producing single-diameter alginate microbeads (MAB).

As described above, conventionally, it has been difficult to produce microbeads of alginic acid having a micro size and a uniform diameter.
The present invention is, in view of the above circumstances, and an object thereof is to provide a manufacturing method and a manufacturing equipment of a single diameter alginate microbeads can be produced a homogeneous micro beads using microfluidics.

In order to achieve the above object, the present invention provides
[1] In a method for producing single-diameter alginate microbeads, a sodium alginate solution containing an oil that is a continuous phase and a sodium alginate solution that contains a substance that is insoluble in an aqueous solution of ph7 or more that is a dispersed phase is crossed from A droplet composed of sodium alginate containing the substance and an oil containing amphiphilic molecules and acetic acid are merged in a two-way merging chamber to produce single-diameter alginate microbeads and mixed. And promoting the gelation of the microbeads in the reaction channel.

[2] The method for producing single-diameter alginate microbeads according to [1] above, wherein the substance is a compound composed of calcium.
[3] The method for producing single-diameter alginate microbeads according to [1] above, wherein the substance is a compound composed of barium.
[4] The method for producing single-diameter alginate microbeads according to [1], wherein the droplets made of sodium alginate are generated by intersecting the continuous phase and the dispersed phase at a T-shaped intersection. And

[5] The method for producing single-diameter alginate microbeads according to [1] above, wherein when the single-diameter alginate microbeads are produced, cells are introduced into the single-diameter alginate microbeads and encapsulated. And
[6] The method for producing single-diameter alginate microbeads according to [ 5 ] above, wherein the cells are cancerous liver cells.

[7] In the apparatus for producing single-diameter alginate microbeads, a first microchannel for flowing oil as a continuous phase and a second alginate solution containing a substance that is insoluble in an aqueous solution of ph7 or more as a dispersed phase. A microchannel, an intersection where the first microchannel and the second microchannel intersect, a droplet made of sodium alginate containing the substance formed at the intersection, an amphipathic molecule and acetic acid. A third microchannel through which the oil containing flows, a two-way merging chamber in which the first microchannel and the third microchannel merge to produce single-diameter alginate microbeads, and the two-way merging chamber communicated and, mixing and reaction to gel micro beads formed in the two-path joint chamber Characterized by comprising a Yan'neru.

[8] The apparatus for producing single-diameter alginate microbeads according to [7] above, wherein the substance is a compound composed of calcium.
[9] The apparatus for producing single-diameter alginate microbeads according to [7] above, wherein the substance is a compound composed of barium.
[10] The apparatus for producing single-diameter alginate microbeads according to [7] above, wherein the intersection is a T-shaped intersection.

  According to the present invention, alginate beads having a uniform size can be produced on a micro scale. It is possible to introduce cells into this, or to array the microbeads on a chip.

Sodium alginate solution containing a manufacturing method and a manufacturing apparatus of a single diameter alginate microbeads, a first microchannel to flow the oil is a continuous phase, a substance which is insoluble in ph7 more aqueous solutions of the dispersed phase of the present invention A second microchannel through which the first microchannel and the second microchannel intersect, a droplet made of sodium alginate containing the substance formed at the intersection, an amphiphile A third microchannel through which an oil containing a sex molecule and acetic acid flows, a two-way confluence chamber in which the first microchannel and the third microchannel merge to form a single-diameter alginate microbead; The microbead formed in the two-way merge chamber is gelled in communication with the two-way merge chamber. Comprising a coupling and reaction channels.

Hereinafter, embodiments of the present invention will be described in detail.
FIG. 1 is a schematic view of a MAB manufacturing apparatus showing an embodiment of the present invention.
In this figure, 1 is a first microchannel (for example, having a diameter of 150 μm and a height of 55 μm) for flowing oil 2 as a continuous phase, and 3 is a flow of sodium alginate solution 4 containing CaCO ₃ powder as a dispersed phase. 2 microchannels (for example, the diameter is 50 μm, the height is 55 μm), 5 is a T-shaped intersection where the first microchannel 1 and the second microchannel 3 intersect, and 6 is the T-shaped intersection 5. A droplet made of sodium alginate containing CaCO ₃ powder to be formed, 7 is a third microchannel through which oil 8 containing lecithin and acetic acid flows, and 9 is the first microchannel 1 and the first microchannel. two-way merging chamber 3 microchannel 7 are merged, microbeads 10 are formed by a two-path joint chamber 9 (mu droplets), 1 mixing and reaction channels communicating with the two-way merging chamber 9, 12 is a microbead advanced gelation (mu droplets).

Here, when a sodium alginate solution 4 containing insoluble calcium carbonate particles is caused to flow in the flow of oil 2 through a T-shaped intersection 5, droplets 6 are generated. An oil 8 containing lecithin and acetic acid is then added downstream from the third microchannel 7. Then, in the mixing and reaction channel 11 consisting of tortuous channels, the mixing of the acidic oil, ie the oil 8 containing lecithin and acetic acid, and the oil 2 flowing in the main stream is promoted. The droplet 6 made of sodium alginate containing CaCO ₃ powder has a reduced pH (ph) value, and when Ca ²⁺ (calcium ions) are combined, gelation occurs, and a calcium alginate gel is formed downstream. That is, due to the decrease in pH value, Ca ²⁺ is released from the insoluble calcium complex, and gelation occurs. Subsequently, the MAB is captured by the microtube and further separated into an aqueous calcium chloride aqueous solution by centrifugal force for further gelation.

Using this single diameter alginate microbead manufacturing device, the cell can be encapsulated in MAB simply by adding the cell to the sodium alginate solution. In this case, however, the solution needs to be adjusted to the osmotic pressure using saline instead of water.
In the above embodiment, a sodium alginate solution containing insoluble CaCO ₃ powder as a dispersed phase has been described. However, any substance that does not dissolve in an aqueous solution of ph7 or higher may be used. For example, barium compound powder may be used.

Further, the intersection is not limited to a T-shaped intersection, and may be flow focusing.
Furthermore, the surfactant is not limited to lecithin, and may be other as long as it is an amphiphilic molecule.
Figure 2 is a MAB drawing-substitute photographs of illustrating an embodiment of the present invention, prior to FIG. 2 (a) is completely gelled oil, FIG. 2 (b) after being fully gelled The figure which shows the state in water, FIG. 3 is a figure which shows the diameter distribution of MAB before and behind gelatinizing completely concerning this invention.

The coefficient of variation (CV) is defined as the ratio of the standard deviation of diameters to the average diameter. The average diameter and coefficient of variation (CV) were 116.93 μm and 1.9% before complete gelation, and 81.91 μm and 2.7% after complete gelation, respectively. 8.9% of the produced MAB was not spherical and was not included in the measurement. Droplet formation using a T-shaped intersection has made it possible to produce single diameter sodium alginate droplets. By adding a surfactant (lecithin), unnecessary adhesion between the formed droplets was prevented, and the single diameter of the microbead could be maintained while the microbead gradually gelled. In this way, the gel hardened before separation into an aqueous phase by centrifugation. As a result, the microbeads (MAB) were spherical and had a small size distribution. By fine tuning parameters such as Ca ²⁺ concentration and pH, defects are reduced or eliminated.

The diameter of the formed microbeads was larger than the channel depth (55 μm). This is because gelation is a slow process and the sodium alginate droplets do not cure completely in the apparatus. These droplets exit the device and take a spherical shape in the microtube before fully gelling.
As is apparent from FIG. 3, the diameter decreases by 29.9% during gelation.

FIG. 4 is a diagram showing a Hep G2 cell (human hepatoma-derived cell) encapsulated in MAB according to the present invention.
In this figure, a Hep G2 cell 15 successfully encapsulated in MAB 16 is shown. Thus, by combining the internal gelation method and the T-shaped intersection drop generation method, a single diameter MAB 16 was generated, and the Hep G2 cell 15 was successfully encapsulated in the MAB 16 . The number of cells encapsulated in MAB can be controlled by adjusting the cell density in sodium alginate.

FIG. 5 is a schematic diagram of a microbead (μ droplet) capturing device (microarray) showing a reference example of the present invention.
In this figure, 21 is a microbead (μ droplet), 22 is a linear channel, 23 is a square wave channel, and 24 is a constricted region formed in the linear channel 22. In FIG. 5, a thick arrow indicates a main stream, and a small arrow indicates a substream.

Therefore, the microbead (μ droplet) capturing device includes a square-wave channel 23 formed so as to intersect the linear channel 22, and the microbead (μ droplet) 21 is captured by the linear channel. This is done in a stenosis region 24 along 22. When the microbead (μ droplet) 21 is not captured and the constriction region 24 is empty, the resistance to the flow from the linear channel 22 to the constriction region 24 is more than the resistance to the flow along the square-wave channel 23. The low, that is, the main stream (thick arrow) flows along the constricted region 24 from the linear channel 22, and the microbead (μ droplet) 21 easily moves from the linear channel 22 to the constricted region 24. Thus, microbeads (mu droplets) 21 in the stream is captured confinement region 24 by the main stream (thick arrow) (trapping mode). Once the microbeads (μ droplets) 21 are captured and the stenosis region 24 is filled, the resistance to flow from the straight channel 22 to the stenosis region 24 is greatly increased, and the main stream is along the square-wave channel 23. The direction is changed. Accordingly, the next microbead (μ droplet) is carried along the square-wave channel 23 (bypass mode), bypassing the buried constriction region 24. The widths of the straight channel 22 and the constricted region 24 are 150 μm and 35 μm, respectively. Each channel height is 85 μm.

In this way, the microbeads (μ droplets) 21 block the constricted region 24 formed in the linear channel 22 in the flowing order, and as a result, the microbeads (μ droplets) 21 are arranged in an array. can do.
Next, production of the above-described apparatus will be described.
Due to the advantages of being capable of rapid prototyping, ease of fabrication, and most importantly biocompatibility, elastomer PDMS (manufactured by Dow Corning, Inc.) can be used to fabricate both MAB and microarray devices. Soft lithography using a silicone elastomer (Sylgard) is selected. First, a SU-8 mold is formed on a silicon wafer using a normal lithography technique. Next, a PDMS film is formed from a SU-8 master mold. Access holes for inlets and outlets are opened in the PDMS plate. Another PDMS plate is produced by curing PDMS in a Petri dish. Finally, the surfaces of the two PDMS plates are treated with oxygen plasma and permanently bonded.

Next, materials used in this apparatus will be described.
In this apparatus, sodium alginate and corn oil, with lecithin extracted from soybean (manufactured by Wako Pure Chemical Industries, Ltd.). Furthermore, calcium chloride (CaCl ₂ ), sodium chloride (NaCl), and Tween 20 (manufactured by Kanto Chemical Co., Inc.) were used. In addition, acetic acid (manufactured by Nacalai Tesque) was used. Calcium carbonate (CaCo ₃ ) powder is available from YHNANO co. , Ltd., Ltd. Provided by. All chemicals and reagents were used as received and without further purification. The ultrapure water used in all experiments was obtained from a Millipore system having a specific resistance of 18 MΩ · cm.

Next, the MAB generation procedure will be described.
A syringe pump (not shown) was used to control the flow rate of all phases. As the continuous phase, corn oil (main stream) and corn oil containing lecithin (2 wt%) and acetic acid (0.67 μl / ml) were used. A sodium alginate (2 wt%) solution containing CaCO ₃ particles was used as the dispersed phase. The flow rates of mainstream corn oil, corn oil acidified with acetic acid, and alginate suspension with CaCO ₃ were 0.25 ml / h, 0.25 ml / h, and 0.018 ml / h, respectively.

The produced microbeads were captured in a microtube. The microtube was centrifuged and the supernatant was removed. The microbeads were gently pipetted and washed with a CaCl ₂ (2 wt%) solution (1 wt% Tween 20). The mixture was then centrifuged again. This washing process was repeated two more times. MAB photographs were taken with a VH-Z450 high magnification zoom lens (450X) and a Keyence VHX-100 digital microscope system. Diameter was measured with Keyence image software.

Next, generation of MAB that encapsulates a cell will be described.
Similar to the MAB procedure, but to maintain cell viability, all solutions had to be adjusted for osmotic pressure. A sodium alginate (2 wt%) solution was prepared using a NaCl (0.9 wt%) solution instead of water. For the cleaning, a CaCl ₂ (155 mM) solution (1 wt% Tween 20) was used. Medium was used for the final wash.

Next, capture of microbeads on the microarray will be described.
The MAB prepared by the above procedure was introduced into the microarray using a syringe pump. Experiments were visualized using a CCD camera (Hamamatsu C5405-05) mounted on an inverted Nikon microscope (Nicon Eclipse, TE300).
6 is a MAB drawing substitute photograph of which is captured in capture arrays of microbeads showing an experimental example of the exemplary embodiment of the present invention (mu droplets).

Here, the microbead (μ droplet) capture array can capture 100 droplets in series.
6A shows a state in which microbeads (μ droplets) are captured on a microbead (μ droplet) capture array, FIG. 6B shows a superimposed image in bypass mode, and FIG. 6C. Indicates superimposed images in the capture mode.

As is clear from this figure, the microbeads (μ droplets) were not captured in the constricted region where other microbeads (μ droplets) were already present. Since the efficiency of this capture is almost 100%, it is suitable for capturing and arraying a small number of microbeads (μ droplets). Regions enclosed by squares in FIG. 6A indicate the bypass mode and the capture mode , respectively. This device was able to immobilize microbeads (μ droplets), and a microarray was formed by continuously connecting these captures.

  Thus, the microbeads were fixed and arrayed by using a continuous microfluidic trap. The ability to generate single diameter MAB can facilitate the use of alginic acid for both cellomics research and therapeutic purposes. The proposed passive microfluidic trap is simple to operate, robust and highly efficient. Drugs / pharmaceuticals can be easily introduced upstream without breaking the trapped microbeads encapsulating the cell, making it a useful tool for cell assay. Furthermore, by encapsulating the cells in alginic acid, not only non-adherent cells but also more difficult adherent cells can be handled.

According to the present invention, by introducing a drug or the like into microbeads having a uniform diameter, the amount of the drug mixed inside can be adjusted. In addition, if cells that release or decompose beneficial substances such as insulin are encapsulated and introduced into the body, they can be utilized while preventing an immune reaction when placed in the body.
Hereinafter, the pressure drop in the microchannel will be described.

The following equation (1) was obtained by determining the pressure drop or pressure difference in the microchannel using the Darcy-Weisbach equation and solving the continuity equation and momentum equation for the problem of the Hagen-Poiseille flow.
Δp = (fL / D) · (ρV 2/2) ... (1)
Where Δp is the pressure difference, f is the Darcy friction coefficient, L is the channel length, D is the fluid diameter, ρ is the fluid density, and V is the average velocity of the fluid.

The fluid diameter D of the microchannel is
D = 4A / P (2)
Here, A is the cross-sectional area of the microchannel, and P is the wet side of the microchannel.
The cross-sectional area and rim of the rectangular channel (W is the width, H is the height) having the width shown in FIG. 7 are shown as follows.

A = WH (3)
P = 2 (W + H) (4)
The Darcy friction coefficient f is related to the Reynolds number Re and the aspect ratio α. The aspect ratio α is defined by either height / width or width / height, and 0 ≦ α ≦ 1. The product of the Darcy friction coefficient f and the Reynolds number Re is a constant depending on the aspect ratio, and
f · Re = C (α) (5)
Where C (α) is a constant C which is a function of α, and Re is Re = ρVD / μ (6)
However, μ is fluid viscosity.

Next, laminar friction coefficients of rectangular channels having various aspect ratios will be described.
Table 1 shows the solutions given by White (see Non-Patent Document 9 above).

Substituting Equations (5) and (6) into Equation (1) yields Equation (7) below.
Δp = [C (α) / 2)] · (μLV / D ² ) (7)
The average velocity V of the fluid is V = Q / A (8)
Where Q is the volumetric flow rate.

Substituting Equation (2) and Equation (8) into Equation (7) yields Equation (9) below.
Δp = [C (α) / 32)] · (μLQP ² / A ³ ) (9)
FIG. 8 is a detailed explanatory view of a micro droplet trap showing a reference example of the present invention.
As described above, the capture of the micro droplet is performed by the square wave channel (loop channel) formed in the linear channel. Microdroplet traps are made in a constricted region along the straight channel, and if the trap is empty, the fluid resistance along the straight channel is lower than the fluid resistance along the loop channel. The beads in the stream are carried and captured by the main stream (capture mode). When trapped, the fluid resistance increases rapidly along the straight channel, and its main stream is redirected to the loop channel, followed by the beads along the loop channel by bypassing the buried trap. Carried.

Here, FIG. 8B shows a simplified circuit diagram of the capture. Two intersections A and B in FIG. 8B correspond to the intersections marked by X in FIG. 8A. The fluid in the intersection A flows to the intersection B via the channel 1 or the channel 2. If a small loss due to bending, expansion of width, etc. is ignored, the pressure difference between the channel 1 and the channel 2 is calculated by the equation (9). Since both channels have the same pressure drop, the following equation (10) is obtained assuming that both equations are equal.

Q ₁ / Q ₂ = [C ₂ (α ₂ ) / C ₁ (α ₁ )] · (L ₂ / L ₁ ) ·
(P ₂ / P ₁ ) ² · (P ₂ / P ₁ ) ² · (A ₁ / A ₂ ) ³ (10)
Here, the subscripts 1 and 2 indicate the flow channel 1 and the flow channel 2, respectively. Note that the channel ₁ has the length of the channel with the width of the length L ₁ narrowed in order to simplify the analysis. This is effective because most pressure drops occur along narrow channels.

The relationship between the area and the surrounding area is as follows:
A ₁ = W ₁ H (11)
A ₂ = W ₂ H (12)
P ₁ = 2 (W ₁ + H) (13)
P ₂ = 2 (W ₂ + H) (14)
Note that the height H is the same in both channels.

Substituting the above relationship into equation (10) yields the following equation (15).
Q ₁ / Q ₂ = [C ₂ (α ₂ ) / C ₁ (α ₁ )] · (L ₂ / L ₁ ) ·
[(W ₂ + H) / (W ₁ + H)] ² · (W ₁ / W ₂ ) ³ (15)
In order for capture to work, the volumetric flow along channel 1 must be greater than the volumetric flow along channel 2. That is,
Q ₁ / Q ₂ ≧ 1
Or [C ₂ (α ₂ ) / C ₁ (α ₁ )] • (L ₂ / L ₁ ) •
[(W ₂ + H) / (W ₁ + H)] ² · (W ₁ / W ₂ ) ³ ≧ 1 (16)
It is.

Note that the above equation does not include a flow rate term. In other words, correctly designed trapping takes place at all speeds in laminar flow.
FIG. 9 is a diagram showing beads captured in a micro droplet capturing apparatus designed with a volume flow ratio Q ₁ / Q ₂ of 3.66 according to a reference example of the present invention.
In this micro-droplet device 31, the volume flow rate ratio Q ₁ / Q ₂ is greater than 1, so that the main stream is along the straight channel 32 and the beads (15 μm) 34 are connected to the straight channel 32. It was captured in the stenosis region 33 and arranged in an array.

In addition, although it tried to capture in the micro droplet capturing apparatus designed with the volume flow ratio Q ₁ / Q ₂ of 0.76, the volume flow ratio Q ₁ / Q ₂ is smaller than 1, and as a result, The main stream was along the loop channel (square wave channel) and could not be captured.
FIG. 10 is a view showing a captured bead releasing (release) apparatus (part 1) showing a reference example of the present invention, and FIG. 10 (a) is a view showing a captured state of the bead, FIG. ) Is a diagram showing a state in which the captured beads are released.

As shown in FIG. 10 (a), the end of the confinement region 42 of the straight tea down channel 41 aluminum 43 is patterned, the bead 44 is trapped in the narrowed region 42. Therefore, as shown in FIG. 10B, a laser (for example, output 1.5 W) 45 is irradiated to the aluminum 43 at the end of the constricted region 42. Then, bubbles 46 are generated at the end of the stenosis region 42, and the beads 44 captured in the stenosis region 42 can be released from the stenosis region 42.

In the above embodiment, the patterned aluminum 43 is irradiated with the laser 45, but the bubble 46 may be generated by irradiating the target substrate with the laser at the end of the constricted region. I can do it.
In this manner, the beads once captured are released by generating bubbles by heating the end of the constriction region using a laser. In other words, the beads once captured can be rearranged freely.

In addition, a device that uses dielectrophoresis to classify beads and cells in a microsystem has been proposed (see Non-Patent Document 10 above).
FIG. 11 is a view showing the operation of the captured bead release device (part 2) showing a reference example of the present invention, FIG. 12 is a view showing the bead release device and the operation thereof, and FIG. FIG. 12 (b) shows a state where a bead (cell) is captured, FIG. 12 (b) shows a state where a reagent is blown, FIG. 12 (c) shows a state where a bead (cell) is selected, and FIG. 12 (d) shows a potential applied to an electrode. FIG. 12E shows a state where the beads (cells) are released.

As shown in FIG. 11, the first electrode 51 has a right triangle shape, and has an oblique side 52, a horizontal side 53, and a vertical side 54. On the other hand, the second electrode 55 also has a right triangle shape, and has an oblique side 56, a horizontal side 57, and a vertical side 58. The oblique side 52 of the first electrode 51 and the vertical side 58 of the second electrode 55 face each other.
Therefore, as shown in FIG. 11A, when a positive potential is applied to the first electrode 51 and a negative potential is applied to the second electrode 55, the lower end where the hypotenuse 52 and the horizontal side 53 of the first electrode 51 cross each other. Since the portion 59 is pointed, and the vertical side 58 of the second electrode 55 is flat, an unequal electric field is generated between the first electrode 51 and the second electrode 55. That is, an electric field with a large inclination is generated at the lower tip portion 59, and an electric field with a small inclination is generated on the vertical side 58 of the second electrode 55. As described above, since an unequal electric field is generated between the first electrode 51 and the second electrode 55, beads (cells) 60 are arranged in the vicinity of the lower end portion 59 as shown in FIG. In this case, the dielectrophoretic force 61 A acts as shown by an arrow, and the bead (cell) 60 generates an electric field with a small inclination from the lower tip 59 where an electric field with a large inclination is generated. The suction is performed to the vertical side 58 of the second electrode 55.

Such means for generating a dielectrophoretic force can be used in the above-described bead (cell) capturing device to release it from the capturing state.
This will be specifically described below.
First, as shown in FIG. 12A, beads (cells) 65 are captured by a microbead capturing array device. In the capture array device, the electrode 64 composed of the first electrode and the second electrode described above is disposed in the narrowed region 63 of the linear channel 61.

Next, as shown in FIG. 12B, the reagent 66 is introduced into the capture array device of the bead (cell) 65, and the reaction of the bead (cell) 65 with respect to the reagent 66 is observed.
Next, as shown in FIG. 12 (c), when there is a desired bead (cell) 65 'to be released, the electrode 64' is selected to release the bead (cell) 65 'and the electrode 64' is selected. Apply an arbitrary voltage to

Next, as shown in FIG. 12 (d), since an unequal electric field is generated between the electrodes 64 'by applying a voltage to the electrodes 64', the beads (cells) 65 'are linearly moved by the dielectrophoretic force. From the constricted region 63 of the channel 61.
Next, as shown in FIG. 12 (e), the released beads (cells) 65 ′ ride on the main stream 67 and flow through the square-wave channel 62.

The released beads (cells) 65 'are transported from the capture array device and collected for further analysis. The potential for generating the dielectrophoretic force is 5-15V.
In addition, this invention is not limited to the said Example, Based on the meaning of this invention, a various deformation | transformation is possible and these are not excluded from the scope of the present invention.

The present invention can be used as a method for producing single-diameter alginate microbeads and a production apparatus thereof.

It is a schematic diagram of the MAB manufacturing apparatus which shows the Example of this invention. A MAB drawing substitute photograph of showing an embodiment of the present invention. It is a diagram showing a diameter distribution of the MAB before and after the complete gelling of an embodiment of the present invention. During MAB according to the present invention is a diagram showing an encapsulated Hep G2 cells. It is a schematic diagram of a microbead (μ droplet) capturing device (microarray) showing a reference example of the present invention. Substituting a drawing photographs of MAB captured in capture arrays of microbeads showing an experimental example of the reference example (mu droplets) of the present invention. It is sectional drawing of the rectangular microchannel of the reference example of this invention. It is detailed explanatory drawing of the micro droplet trap which shows the reference example of this invention. Volumetric flow rate ratio Q ₁ / Q ₂ showing a reference example of the present invention is a diagram showing the beads trapped in the trapping device designed microdroplets 3.66. It is a figure which shows the releasing (release) apparatus (the 1) of the capture | acquired bead which shows the reference example of this invention. It is a figure which shows operation | movement of the releasing apparatus (the 2) of the capture | acquired bead which shows the reference example of this invention. It is a figure which shows the releasing apparatus (the 2) of the capture | acquired bead which shows the reference example of this invention, and its operation | movement.

1 First microchannel 2 Oil (continuous phase)
3 Second microchannel 4 Sodium alginate solution containing CaCO ₃ powder (dispersed phase)
5 T-shaped intersection 6 Droplet made of sodium alginate containing CaCO ₃ powder 7 Third microchannel 8 Oil containing lecithin and acetic acid 9 Two-way merge chamber 10 Microbeads formed in the two-way merge chamber (μ solution drop)
11 Mixing and reaction channel 12 Micro-beads with advanced gelation (μ droplet)
15 Hep G2 cell
16 , 21 Micro beads (μ droplet)
22, 32, 41, 61 Linear channel 23, 62 Square wave channel 24, 33, 42, 63 Constriction region formed in linear channel 31 Micro droplet device 34 Bead (15 μm)
43 Aluminum 44 Bead 45 Laser 46 Bubble 51 First electrode 52, 56 Oblique side 53, 57 Horizontal side 54, 58 Vertical side 55 Second electrode 59 Lower tip 60, 65 Bead (cell)
61 A Dielectrophoretic force 64 Electrode composed of first electrode and second electrode 64 ′ Electrode to apply voltage 65 ′ Desired bead (cell) to be released
66 Reagents 67 Mainstream

Claims (10)

(A) Crossing an oil that is a continuous phase and a sodium alginate solution containing a substance that is insoluble in an aqueous solution of ph7 or more that is a dispersed phase to form droplets made of sodium alginate containing the substance,
(B) A droplet made of sodium alginate containing the substance and an oil containing an amphiphilic molecule and acetic acid are joined in a two-way merging chamber to produce single diameter alginate microbeads,
(C) A method for producing single-diameter alginate microbeads, which promotes gelation of the microbeads in a mixing and reaction channel.   2. The method for producing single-diameter alginate microbeads according to claim 1, wherein the substance is a compound comprising calcium.   The method for producing single-diameter alginate microbeads according to claim 1, wherein the substance is a compound comprising barium.   The method for producing single-diameter alginate microbeads according to claim 1, wherein the droplets made of sodium alginate are generated by intersecting the continuous phase and the dispersed phase at a T-shaped intersection. Method for producing diameter alginate microbeads.   The method for producing single-diameter alginate microbeads according to claim 1, wherein when the single-diameter alginate microbeads are produced, cells are introduced into the single-diameter alginate microbeads and encapsulated. Method for producing diameter alginate microbeads. 6. The method for producing single-diameter alginate microbeads according to claim 5 , wherein the cells are cancerous liver cells. (A) a first microchannel through which oil that is a continuous phase flows;
(B) a second microchannel in which a sodium alginate solution containing a substance that is not soluble in an aqueous solution of ph7 or higher that is a dispersed phase is passed;
(C) an intersection where the first microchannel and the second microchannel intersect;
(D) a droplet made of sodium alginate containing the substance formed at the intersection, a third microchannel through which an oil containing an amphiphilic molecule and acetic acid flows;
(E) a two-way merge chamber in which the first microchannel and the third microchannel merge to produce single diameter alginate microbeads;
(F) An apparatus for producing single-diameter alginate microbeads comprising a mixing and reaction channel that communicates with the two-way merging chamber and gels the microbeads formed in the two-way merging chamber. .   8. The apparatus for producing single-diameter alginate microbeads according to claim 7, wherein the substance is a compound made of calcium.   8. The apparatus for producing single-diameter alginate microbeads according to claim 7, wherein the substance is a compound made of barium.   8. The apparatus for producing single diameter alginate microbeads according to claim 7, wherein the intersection is a T-shaped intersection.