JP2009125635A - Resettable apparatus for arrangement of microdrop - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a resettable apparatus for arrangement of microdrops which arranges microdrops by forming microdrops into arrays through two or more constricted areas (beds) arranged horizontally in the head, captures microdrops easily, releases (discharges) microdrops reliably and enables high-speed operation. <P>SOLUTION: The resettable apparatus for arrangement of microdrops keeps two or more constricted areas 4 arranged horizontally and communicating with a passage 3 through which a liquid 1 flows and arranges microdrops 2 in the form of arrays on a chip. It has a micro-passage 5 communicating with the rear part of the constricted areas 4, microdrops 2 to be captured in the constricted areas 4 and a control member (obstacle) 7, or an obstacle, arranged on the side of the outlet 6 of the micro-passage 5, and the captured microdrops 2 are released all at once from the constricted areas 4 under the action of the control member (obstacle) 7 produced through a reversed operation of the liquid 1 flowing through the passage 3. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention is not only applicable to biotechnology fields such as microchemical engineering, drug delivery, cell culture, and regenerative medical engineering, but also to resettable microarrays that are expected to be applied to industries that place and use microbeads such as the display industry. The present invention relates to an apparatus for arranging droplets.

  In cell research, drug selection, and detection / diagnostic applications, integrated manipulation systems that can continuously observe microdroplets such as biological cells under chemical stimulation are in great demand ( Non-patent document 1 below). Such a system needs to have many functions such as microdroplet transport and fixation, reagent injection, reaction observation, selected microdroplet release and the like.

  The inventors of the present application have achieved all of the functions described above through a combination of fluid dynamics and an optical approach (see Non-Patent Document 2 below). This system makes it possible to arrange microdroplets in a short time, as well as release selected droplets for microarray applications.

FIG. 6 is a schematic diagram showing a state of releasing (selecting) a microdroplet captured using such a conventional optical approach. The laser 103 is focused on a 1 μm square aluminum plate 102 located on the right side of the chamber 101 to heat the aluminum plate 102 and the solution around it. When the temperature of the aluminum plate 102 reaches the boiling point of the surrounding solution, bubbles 105 are generated in the chamber 101. The bubble 105 pushes the trapped microdroplet 104 into the main flow path, and the microdroplet 104 is released (selected).
Robinson WH, DiGennaro C, Huber W, Haab BB, Kamachi M, Dean EJ, Fournel S, Fong D, Genovese MC, Neumann de Vegvar HE, et al. , "Autoantigen microarrays for multiple plexation of autoantibody re- parations", Nature Medicine, vol. 8, pp. 295-301, 2002 W. Tan, S.M. Takeuchi, “A Trap-and-Release Integrated Microfluidic System For Dynamic Microarray Applications”, PNAS, vol. 104, no. 4, pp. 1146-11151 2007 W. Tan, S.M. Takeuchi “An Optical Retrieval Microfluidic System for Microarray Applications”, in Digest Tech. Papers μTAS'06 Conference, Tokyo, November 5-9, 2006, pp. 509-511 C. Amador, A .; Gavrilidis, P.M. Angeli, “Flow Distribution In Different Microreactor Scale-out Geometrics And The Effect of Manufacturing Tolerances and Channel Blockage.” 101, pp 379-390, 2004

  However, the conventional system described above did not have the function of releasing all captured microdroplets at once and resetting the device. As reported in Non-Patent Document 3 above, it is possible to reset such a device by releasing all the microdroplets captured using the optical release microfluidic method one by one. However, this method is time consuming and impractical for high density (> 1000) microarrays. Cell research and drug selection require the acquisition of a huge number of data to draw meaningful conclusions. For this purpose, a resettable arrangement device for repeated use is desired.

  FIG. 7 is a diagram for explaining the problems of the prior art.

In FIG. 7A, the micro droplet 201 is captured in the constriction region 202 by the flow of the liquid 200 in the forward direction. When the liquid 200 is introduced in the forward direction, the flow is split into two flows. A trapped flow whose flow rate is shown as Q1 and a main flow whose flow rate is shown as Q2. The flow rate ratio (Q1 / Q2) is determined only by the channel structure. And it does not change with the material of the fluid introduce | transduced, and its flow volume (refer the said nonpatent literature 4). If the flow ratio (Q1 / Q2) is greater than 1, the center of the mass of the microdroplet is contained in the trapping flow region and the microdroplet is trapped in the constriction region. Therefore, as shown in FIG. 7B, when the micro droplet 201 captured in the constriction region 202 is released by a backflow operation for introducing the liquid 200 in the reverse direction, The micro droplet 202 is captured at the outlet 204 of the communicating micro passage 203. That is, when the flow rate ratio Q ₁ / Q ₂ > 1, the micro droplet 201 captured as shown in FIG. 7A is released by a back-flow operation that reversely flows the liquid 200 as shown in FIG. 7B. There was a problem that it was impossible to do.

  In view of the above situation, the present invention captures microdroplets in a resettable microdroplet arrangement device in which a plurality of constricted regions (beds) are arranged in the horizontal direction and microdroplets are arranged in an array. It is an object of the present invention to provide an array arrangement of resettable micro droplets that is easy to release, can ensure the release (release) of micro droplets, and can increase the speed.

In order to achieve the above object, the present invention provides
[1] In a resettable microdroplet arrangement device in which a plurality of constricted regions communicating with a flow path through which liquid flows are arranged in a horizontal direction and microdroplets are arranged in an array, the devices communicate with the rear of the constricted region. And a control unit disposed on the outlet side of the microchannel, and the control member acts by a backflow operation of the liquid flowing in the channel. The trapped microdroplets are released simultaneously from the constriction region.

  [2] The resettable microdroplet arranging apparatus according to [1], wherein the control member is arranged in an arc shape so as to cover an outlet side of the microchannel, and includes a plurality of rod-shaped members having a circulation space. It is characterized by becoming.

  [3] The resettable microdroplet arranging apparatus according to [1], wherein the control member has a function of guiding microdroplets in the flow path to the narrowed region.

  [4] The resettable microdroplet arranging apparatus according to [1], wherein the control member is a valve mechanism.

  [5] The resettable microdroplet arranging apparatus according to [4], wherein the valve mechanism is opened when the liquid is flowing in a forward direction, and is closed by a backflow operation of the liquid.

  [6] The resettable microdroplet arranging apparatus according to [1], wherein the microdroplets are gel beads.

  [7] The resettable microdroplet arranging device according to [1], wherein the microdroplets are living cells.

  According to the present invention, it is possible to release the trapped microdroplets all at once by performing a back flow operation on a liquid in a single device, and to reset the device simply and at high speed.

  The resettable microdroplet arrangement device of the present invention is a resettable microdroplet in which a plurality of constricted regions communicating with a flow path through which a liquid flows are arranged in a horizontal direction and the microdroplets are arranged in an array. In the arrangement apparatus, the microchannel includes a microchannel communicating behind the stenosis region, a microdroplet trapped in the stenosis region, and a control member disposed on the outlet side of the microchannel, and flows into the channel. The control member acts by a liquid backflow operation to release the trapped microdroplets simultaneously from the constriction region.

  In the present invention, the microdroplet means a microbead, a gel bead, or a living cell such as a cell.

  Hereinafter, embodiments of the present invention will be described in detail.

  FIG. 1 is a schematic view of a main part of a resettable microdroplet arranging apparatus showing an embodiment of the present invention.

As shown in FIG. 1A, the micro droplet 2 is trapped in the constriction region (bed) 4 by the flow of the liquid 1 in the forward direction. In the present invention, the flow rate ratio Q ₁ / Q ₂ is further increased by installing in the flow path 3 that flows through the control member (obstacle) 7 that covers the outlet 6 side of the micro passage 5 that communicates with the rear of the constriction region 4. As shown in FIG. 1B, the microdroplets 2 trapped in the constricted region 4 are released at the same time by a backflow operation for backflowing the liquid 1 as shown in FIG.

The apparatus of the present invention is configured such that even if the flow rate ratio Q ₁ / Q ₂ <1, the control member (obstacle) 7 guides all the micro droplets 2 as shown in FIG. Therefore, the micro droplet 2 can be reliably captured in the constriction region (bed) 4. On the other hand, the micro droplet 1 is guided away from the outlet 6 side of the micro passage 5 communicating with the rear side of the constriction region (bed) 4 by the control member (obstacle) 7 by the back flow operation for causing the liquid 1 to flow back. As a result, when the liquid 1 is operated in a reverse flow, the micro droplet 2 is not captured on the outlet 6 side of the micro passage 5 and is separated from the constriction region (bed) 4. The low flow ratio Q ₁ / Q ₂ in this device is converted to a smaller trapping force (due to a smaller flow through trapping). This is particularly advantageous for capturing sensitive biological cells.

  FIG. 2 is a diagram showing the structure of a control member (obstacle) for capturing and releasing 100 μm micro droplets according to an embodiment of the present invention.

  In this example, an apparatus for manipulating 100 μm microdroplets was designed.

  Here, the width of the flow path 3 is set to 247 μm, and a plurality of 40 μm square is formed in a substantially arc shape so as to cover the outlet 6 side of the micro passage 5 communicating with the rear of the narrowed region (bed) 4 in the flow path 3. A rod-shaped control member 7 is disposed. In addition, although the example arrange | positioned in a substantially circular arc shape was shown here, it is not limited to this, You may make it be another shape, for example, a triangular shape.

  FIG. 3 (a) shows a superimpose image showing a state of capturing a micro droplet by a forward flow of the liquid, and FIG. 3 (b) shows a state of releasing the micro droplet by a back flow operation of the liquid. Yes.

  From these figures, it is possible to better understand the capture of microdroplets and the release of the captured microdroplets.

  FIG. 4 shows the appearance of two experiments performed on a single device designed to manipulate 1000 microdroplets (φ15 μm) showing an embodiment of the present invention.

  First, as shown in FIG. 4 (a), biotin-polystyrene beads and non-biotinylated polystyrene beads as microdroplets are captured in the apparatus, and Alexa 488 is captured. A streptavidin solution was introduced into the flow path. Then, as shown in FIG. 4B, a green fluorescent reaction was observed on the beads by the biotin-streptavidin reaction. Further, biotin-polystyrene beads and non-biotinylated polystyrene beads can be distinguished depending on whether or not the beads show a fluorescence reaction. Therefore, the flow of the Alexa 488 streptavidin solution in the flow path is reversed so that the minute trapped in the stenosis region (bed) 4 of this apparatus is shown in FIG. 4 (c). The beads as droplets were released all at once. Thereafter, the above-described two kinds of new beads were introduced into the apparatus by a forward flow.

  Next, Alexa 546 streptavidin solution was introduced into the reset device. Then, as shown in FIG. 4D, a red fluorescence reaction occurred on the biotin-polystyrene beads by the biotin-streptavidin reaction.

  This simple streptavidin analysis experiment has shown that multiple experiments can be easily performed on a single instrument, saving cost and time.

  The resettable microdroplet array device of the present invention reduces the stress on the trapped microdroplets and in particular directly captures them without affecting the living body of the living cell. Can do. Such an apparatus can be applied to simultaneous selection of a plurality of medicines and medical products having beads and living cells.

  INDUSTRIAL APPLICABILITY The present invention can provide a simple and resettable microdroplet array device that can capture, sort and observe microdroplets (beads, cells, etc.) on a large scale and repeatedly. . In the present invention, the release from the trapped stenosis region is performed by a simple fluid backflow operation. This device is simple to operate and has a strong and high efficiency. 1000 microbeds can be arranged and these can be released in minutes.

  Next, another embodiment of the present invention will be described.

  FIG. 5 is a schematic view of a resettable microdroplet array device using a valve mechanism according to another embodiment of the present invention. FIG. 5A is a diagram in which the valve is released by the flow of liquid in the forward direction. FIG. 5B is a schematic diagram showing a state in which the microdroplet is released by closing the valve by the backflow operation of the liquid.

  In FIG. 5A, a valve 15 is disposed on the outlet 14 side of the micro passage 13 located behind the constriction region (bed) 12 where the liquid 10 communicates with the flow path 11, and the liquid 10 moves in the forward direction. Since the valve 15 is open, the flow rate ratio (Q1 / Q2)> 1, and the micro droplet 16 is trapped in the constriction region 12. On the other hand, in FIG. 5B, the valve 15 disposed on the outlet 14 side of the micro passage 13 located behind the constriction region (bed) 12 is closed by the backflow operation that reverses the flow direction of the liquid 10. In order to inhibit the flow of the flow rate Q1 to the outlet 14 side of the micro passage 13 disposed behind the constriction region 12, the flow rate ratio (Q1 / Q2) <1, and the micro droplet 16 is captured by the constriction region 12. Without being released into the flow path 11.

  By configuring as described above, trapping in the constricted area (bed) is ensured, and the trapped microdroplets can be released all at once by a simple liquid backflow operation, and used repeatedly. It is possible to easily and quickly reset the micro droplets of the arrangement device.

  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 resettable microdroplet array device of the present invention can be effectively used not only in the bio field such as regenerative medical engineering, cell research, and drug discovery but also in the display industry.

It is a schematic diagram of the principal part of the arrangement device of the micro droplet which can be reset showing the example of the present invention. It is a figure which shows the structure of the control member (obstacle) for capturing and releasing a 100 micrometer micro droplet which shows the Example of this invention. It is a superimpose image which shows the mode of the capture | acquisition or simultaneous release of a microdroplet by the advancing direction of liquid, or a backflow operation. It is a figure which shows the mode of two experiments performed with the single apparatus designed in order to operate 1000 microdroplets ((phi) 15micrometer) which shows the Example of this invention. It is the schematic diagram of the arrangement device of the micro droplet which can be reset using the valve mechanism which shows the other Example of this invention. It is a schematic diagram which shows a mode that the micro droplet captured using the conventional optical approach is released (selected). It is a figure explaining the problem of a prior art.

Explanation of symbols

1,10 liquid 2,16 micro droplet 3,11 flow path 4,12 constricted region (bed)
5,13 Micro passage 6,14 Exit of micro passage 7 Control member (obstacle)
15 Valve

Claims (7)

In a resettable microdroplet arrangement device in which a plurality of constricted regions communicating with a flow path through which liquid flows are arranged in a horizontal direction and microdroplets are arranged in an array,
(A) a micro passage communicating with the rear of the stenosis region;
(B) a microdroplet trapped in the constriction region;
(C) a control member disposed on the outlet side of the micro passage,
(D) An array of resettable microdroplets characterized in that the control member acts to release the trapped microdroplets from the constricted region all at once by backflow operation of the liquid flowing in the flow path. apparatus.   2. The resettable micro-droplet arrangement device according to claim 1, wherein the control member is arranged in an arc shape so as to cover the outlet side of the micro-passage, and includes a plurality of rod-like members having a circulation space. A resettable microdroplet array device.   2. The resettable microdroplet arrangement device according to claim 1, wherein the control member has a function of guiding the microdroplets in the flow path to the narrowed region. Droplet array device.   2. The resettable microdroplet arranging device according to claim 1, wherein the control member is a valve mechanism.   5. The resettable microdroplet arrangement device according to claim 4, wherein the valve mechanism opens when the liquid is in a forward flow and closes by a backflow operation of the liquid. Droplet array device.   2. The resettable microdroplet arrangement device according to claim 1, wherein the microdroplets are gel beads.   2. The resettable microdroplet arranging device according to claim 1, wherein the microdroplet is a biological cell.