JP2009097939A - Screening device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To reduce the amount of a reagent (sample) used for reaction inspection or the like for screening in drug development, life science or the like. <P>SOLUTION: First of all, an oil 31 is sucked, and then a target compound 41 is sucked into a pipette 14. The target compound 41 is discharged from the pipette 14, and a droplet 41a of the target compound 41 is arranged on the bottom surface of a container 16 filled with the oil 31. Then, a reaction compound 42 in the pipette 14 is sucked. The reaction compound 42 is discharged from the pipette 14 in the container 16 to form a droplet 42a of the reaction compound 42, and the droplet 42a is brought into contact with the droplet 41a to fuse therewith. Occurrence of a reaction between the droplet 41a and the droplet 42a caused by contact fusion is analyzed in an observation system, and a reacted reaction compound 42 is adopted as a medicine candidate. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a screening apparatus used in fields such as drug discovery and life science.

In drug discovery, a screening assay is performed to search for compounds that react with target substances (proteins, etc.) and cells.
In this screening assay, the reaction is generally analyzed by dispensing into a perforated plate or by a fine route. Samples (reagents) such as compounds and enzymes used in the screening assay are generally expensive, and the number of combinations to be searched for is enormous. Therefore, the screening assay is very expensive. For this reason, in order to reduce the cost of the screening assay, it is important to reduce the amount of sample per reaction test (one reaction test).

  In the current screening system, the volume of the sample per reaction test has been reduced to about 1 to 100 μl, but the development cost of new drugs is still enormous, and a further minute amount of capacity per reaction test is required. Is required.

As a technique for reducing the volume per reaction test, a method of microarraying, a method of arranging microdroplets in an array, and the like have been proposed. For example, in order to facilitate handling of microdroplets when performing microscopic screening, a method of forming droplets at hydrophilic spots on a substrate has been proposed (see Patent Document 1). In addition, an apparatus and a method for arranging micro droplets and screening microorganisms have been proposed (see Patent Document 2).
JP-A-11-304666 JP 2005-137288 A

  The test substance or cell is generally a solution or suspension of a volatile solvent such as water. For this reason, simply reducing the volume of the volume is not practical because the concentration change due to the volatilization of the solvent becomes significant. In order to solve this, a device for controlling the atmosphere, a device for adding a solvent, and the like are required, and the system configuration of the system becomes complicated.

  The internal pressure of the droplet is proportional to the interfacial tension and inversely proportional to the radius of curvature of the droplet. This means that the internal pressure of the droplet changes depending on the radius of curvature thereof, and the internal pressure of a micro droplet having a small radius of curvature becomes very large. For this reason, it is difficult to freely control the volume of the fine droplets.

  An object of the present invention is to provide a screening apparatus that can reduce the volume of a reagent (sample) required for one reaction test.

The first aspect of the screening apparatus of the present invention comprises a droplet array means and a fusion means.
In the droplet array means, a droplet array of the first reagent is formed in a liquid that is incompatible with the first reagent. The fusing means brings the second reagent droplets into contact fusion with the first reagent droplets arranged on the droplet array means. The first reagent is, for example, a target compound such as a protein, and the second reagent is, for example, a compound (reaction compound) that tests whether or not there is a reaction with the first reagent.

  According to the first aspect of the screening apparatus of the present invention, the first reagent droplet formed in the liquid incompatible with the first reagent can be contact-fused with the second reagent droplet. . Therefore, it is possible to search for a second reagent that is a drug candidate substance by using a small amount of the first reagent and a small amount of the second reagent.

The second aspect of the screening apparatus of the present invention further comprises droplet array forming means in addition to the droplet array means and the fusion means.
The droplet array forming means discharges the first reagent in a liquid incompatible with the first reagent to form a droplet of the first reagent, and the droplet array means Arranged above to form a droplet array of the first reagent.

  According to the second aspect of the screening apparatus of the present invention, the predetermined amount of liquid droplets can be easily formed simply by discharging a predetermined amount of the first reagent into a liquid that is incompatible with the first reagent. it can. Therefore, a minute amount of droplets can be formed without precise pressure control.

  According to a third aspect of the screening apparatus of the present invention, in the second aspect, the droplet array forming means is aspirated to suck the first reagent after being filled with a liquid that is incompatible with the first reagent. And the first reagent is ejected from the suction means in a liquid that is incompatible with the first reagent to form droplets of the first reagent, and the droplets are formed into the droplet array means. It is comprised so that the droplet formation control means arrange | positioned may be provided.

  The suction means is, for example, a pipette. In this case, the droplet formation control means, for example, controls the internal pressure of the pipette to cause the pipette to suck the liquid that is incompatible with the first reagent and the first reagent, and from the pipette to the first pipette. The first reagent droplets are formed by discharging the first reagent.

  According to the screening apparatus of the third aspect of the present invention, when the first reagent is sucked into the suction means, the first reagent layer and the liquid layer incompatible with the first reagent are stacked in the suction means. To be accommodated. Therefore, when the first reagent is discharged from the suction means in the liquid that is not compatible with the first reagent, there is a problem even if a liquid that is not compatible with the first reagent is discharged from the suction means in addition to the first reagent. Absent. For this reason, the pressure control at the time of discharging the first reagent from the suction means becomes easier than in the second mode.

  According to a fourth aspect of the screening apparatus of the present invention, in the third aspect, the first reagent layer and a liquid layer incompatible with the first reagent provided on the layer are accommodated. First storage means is further provided. The aspirating means aspirates the first reagent from the first accommodating means.

The liquid that is incompatible with the first reagent is, for example, a material that adheres to the tip of the suction means when the suction means sucks the first reagent from the first storage means.
According to the fourth aspect of the screening apparatus of the present invention, the first reagent can be aspirated from the first accommodating means to the aspirating means. In this case, if a material that adheres to the suction means is used as a liquid that is not compatible with the first reagent, the tip portion of the suction means from which the first reagent has been sucked is a liquid that is not compatible with the first reagent. Can be closed. For this reason, when the first reagent is transported by the suction means to produce the droplet array, it is possible to reduce the rate at which components such as moisture volatilize from the first reagent in the suction means. Become.

  According to a fifth aspect of the screening apparatus of the present invention, in the first aspect, the fusion means is a suction means for sucking the second reagent after being filled with a liquid incompatible with the first reagent, Fusion control means for discharging the second reagent from the suction means and bringing the second reagent droplet into contact fusion with the droplet of the first reagent arranged on the droplet array means Configure to include.

  According to the fifth aspect of the screening apparatus of the present invention, a small amount of the second reagent droplet can be formed without the need for precise pressure control by the same action as in the third aspect. Moreover, the presence or absence of these reactions can be tested using both the first reagent and the second reagent in minute amounts.

  According to a sixth aspect of the screening apparatus of the present invention, there is provided a second containing means for containing the second reagent layer and a liquid layer incompatible with the second reagent provided on the second reagent layer. And further. The aspirating unit aspirates the second reagent from the second storage unit.

The liquid that is incompatible with the second reagent is a material that adheres to the tip of the suction means when the suction means sucks the second reagent from the second storage means.
According to the sixth aspect of the present invention, when the second reagent is transported by suction means for contact fusion with the first reagent, the component of the second reagent is obtained by the same action as in the fourth aspect. The rate at which vaporizes can be reduced.

  According to a seventh aspect of the screening apparatus of the present invention, in the first aspect, the droplet array means includes a component that absorbs a liquid that is incompatible with the first reagent from the droplet of the first reagent. Replenishment means for replenishing a liquid that is incompatible with the first reagent is provided.

  According to the seventh aspect of the screening apparatus of the present invention, the components of the droplets of the first reagent in the droplet array formed in the liquid that is incompatible with the first reagent are in phase with the first reagent. The rate of absorption by the undissolved liquid can be reduced, and the rate at which the volume of the first reagent droplet in the droplet array is reduced can be reduced.

  According to the present invention, when screening is performed, the volume of a reagent (sample) required for one reaction test can be greatly reduced as compared with the conventional case. For this reason, the cost required for screening can be reduced and the reaction time in screening can be shortened.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[principle]
First, the principle of the embodiment of the present invention will be described.
In this embodiment, screening is performed to search for reagent 2 as a drug candidate substance by contacting and fusing the droplet of reagent 2 with the droplet of reagent 1 formed in oil and examining whether or not they react. . This screening is performed according to the following procedure.

(1) Fill a container with oil and form a droplet array of reagent 1 on the bottom of the container.
In order to form this droplet array, a pipette previously filled with oil is used. Specifically, the reagent 1 is sucked into a pipette filled with oil, and the reagent 1 is discharged from the pipette in the oil of the container to form a droplet of the reagent 1 at the tip of the pipette. And this droplet is arrange | positioned on the bottom face of a container. By repeating this series of operations, a droplet array of reagent 1 is produced on the bottom surface of the container. The droplet formation of the reagent 1 will be described in more detail. First, the reagent 1 is sucked into the pipette by a predetermined amount (1fl to 1nl) by negative pressure or capillary (capillary) rise. And the reagent 1 in this pipette is discharged in the oil of a container. Since the reagent 1 and the oil are not dissolved (not compatible) with each other, when the reagent 1 is discharged in the oil, droplets of the predetermined amount of the reagent 1 are formed. In this case, the droplet becomes spherical due to the interfacial tension. In addition, since the upper layer of the reagent 1 is filled with oil in the pipette, there is no problem even if the oil is discharged when the reagent 1 is discharged from the pipette because the oil is the same material as the oil in the container. In this manner, a minute amount (1 fl to 1 nl) of the reagent 1 droplet can be formed without precise pressure control. Then, the droplet array of the reagent 1 can be produced by arranging the droplet of the reagent 1 on the bottom surface of the container. When a predetermined amount of reagent is aspirated into the pipette, if the reagent 1 is aspirated more than the predetermined amount, the reagent 1 is discharged from the pipette with positive pressure, and the amount of reagent 1 in the pipette is set to the predetermined amount. You may make it adjust.

(2) Next, a predetermined amount (1 fl to 1 nl) of Reagent 2 is put into a pipette filled with oil in the same manner as Reagent 1. Then, the reagent 2 is discharged from the pipette in the oil of the container in which the droplet array is formed on the bottom surface to form the predetermined amount of the droplet of the reagent 2. This droplet also becomes spherical due to the interfacial tension. The droplet formation of the reagent 2 can also be formed in a very small volume (1 fl to 1 nl) without requiring precise pressure control for the reasons described above. Then, the droplet of the reagent 2 is brought into contact with the droplet of the reagent 1 in the droplet array. Since both Reagent 1 and Reagent 2 are solutions, they fuse easily. In this manner, the reagent 1 droplet and the reagent 2 droplet are contact-fused, and the presence or absence of the reaction is analyzed to search for the reagent 2 that is a drug candidate.

In this way, it is possible to search for a reagent 2 that is a drug candidate substance by examining the presence or absence of a reaction using a small amount (1 fl to 1 nl) of the reagent 1 and the reagent 2.
By the way, normally, liquids are not completely incompatible with each other. For example, water is absorbed in oil at a rate of several hundred ppm or less. For this reason, the droplet of the aqueous solution in oil shrinks at a speed of 10 to 1000 fl / s under normal temperature and humidity. In this case, by holding the solvent of the reagent 1 solution around the reagent 1 droplet, the reduction rate of the reagent 1 droplet can be reduced. Further, the same effect can be obtained even when oil in which moisture absorption is balanced in advance is used. In this case, the oil is used for the screening work after the moisture absorption is in a balanced state by stirring the water and oil or leaving the oil in the screening work environment.

[System configuration]
FIG. 1 is a diagram showing a system configuration of an embodiment of the screening apparatus of the present invention.
The screening apparatus 10 shown in FIG. 1 includes a control system 11, a pressure controller 12, an XYZ stage 13, a pipette 14, a first XY stage 15, a container 16, a second XY stage 17, a reagent 1 library 18, and a reagent 2 A library 19 and an observation system 20 are provided.

  The control system 11 includes a computer or the like, and controls the entire system while controlling the pressure controller 12, the XYZ stage 13, the first XY stage 15, the second XY stage 17, and the observation system 20.

  The pressure controller 12 controls the internal pressure of the pipette 14 under the control of the control system 11, and causes the pipette 14 to perform the operations of aspirating and discharging the reagent 1 and the reagent 2. The XYZ stage 13 is a device for moving the attached pipette 14 in three directions of the X axis, the Y axis, and the Z axis, and the control of the movement is performed by the control system 11. In FIG. 1, the pipette 14 and the XYZ stage 13 are mounted in an oblique direction, but this is an example, and the pipette 14 and the XYZ stage 13 may be mounted in the vertical direction (vertical direction). The pipette 14 is an instrument used for aspirating and discharging the reagent 1 and the reagent 2 and for bringing the reagent 1 and the reagent 2 into contact fusion. For example, the pipette 14 has a length of 4 mm to 5 mm, an outer diameter of 1 mm, and an inner diameter of 0.5 mm. Moreover, the diameter of the hole provided in the front-end | tip part is 1 micrometer-50 micrometers. The first XY stage 15 is an apparatus for moving the container 16 mounted on the upper surface (stage surface) in the X-axis and Y-axis directions under the control of the control system 11. The container 16 is a container for storing the oil 31. The material of the container 16 is, for example, plastic or glass. Under the control of the control system 11, the second XY stage 17 moves each container 18a of the library 18 of the reagent 1 and each container 19a of the library 19 of the reagent 2 mounted on the upper surface (stage surface) of the X axis and It is a device for moving in the Y-axis direction.

  The library 18 of the reagent 1 is a library of a plurality of types of reagents 1 and includes a plurality of containers 18 a that individually store the various types of reagents 1. Reagent 1 is a target compound such as a protein. The library 2 of the reagent 2 is a library of a plurality of types of reagents 2 and includes a plurality of containers 19 a that individually store the various types of reagents 2. Reagent 2 is a compound (drug candidate substance) for examining the reaction with reagent 1. Although only three containers 18a and 19a are shown in FIG. 1, this is merely an example, and the number of containers 18a and 19a is arbitrarily determined according to the system. As will be described later, in the container 18a, the layer of the reagent 1 is accommodated in the lower layer, and the oil layer is accommodated in the upper layer. Further, in the container 19a, a layer of the reagent 2 is accommodated in the lower layer, and an oil layer is accommodated in the upper layer.

  The observation system 20 includes a light source, a microscope, a camera, and the like, and photographs a state change when the droplet of the reagent 1 and the droplet of the reagent 2 are fused together. Then, the captured image is transmitted to the control system 11. The control system 11 analyzes the image received from the observation system 20, and based on the change in the state of the reagent 1 and the reagent 2, the reaction result of the reagent 1 and the reagent 2 (presence / absence of reaction) is displayed on the monitor screen of the display 11a. To display. The presence / absence of the reaction between the reagent 1 and the reagent 2 is determined by, for example, the presence or absence of fluorescence.

  FIG. 2 is a diagram showing a signal waveform for controlling the internal pressure of the pipette 14 applied to the pressure controller 12 by the control system 11. In the graph of FIG. 2, the horizontal axis represents time and the vertical axis represents pressure. The control system 11 controls the internal pressure of the pipette 14 by outputting a pulse waveform signal as shown in FIG. 2 to the pressure controller 12.

[Screening procedure]
FIG. 3 is a diagram showing a basic procedure of a screening technique using the screening apparatus 10 of the present embodiment. The basic procedure of the screening method of this embodiment will be described with reference to FIG. In FIG. 3, the pipette 14 is vertically placed in the container 16. However, as shown in FIG. 1, the pipette 14 may be inclined and placed in the container 16. The same applies to the drawings described later.

(1) The oil 31 is filled in the container 16 placed on the first XY stage 15 (not shown). The oil 31 is oil that does not emit light, such as silicon oil and paraffin oil.
(2) The reagent 18 library 18 and the reagent 2 library 19 are set on the second XY stage 17. In this example, reagent 1 is a target compound (protein) and reagent 2 is a reaction compound (drug candidate substance).
(3) The pressure controller 12 controls the internal pressure of the pipette 14 to suck the oil 31 in the container 16 into the pipette 14.
(4) The internal pressure of the pipette 14 is controlled by the pressure controller 12, and the target compound 41 (reagent 1) in the container 18a is sucked into the pipette 14 filled with the oil 31.
(5) The internal pressure of the pipette 14 is controlled by the pressure controller 12, the target compound 41 is discharged from the pipette 14, and the target compound sucked into the pipette 14 on the bottom surface of the container 16 filled with the oil 31 Forty-one droplets 41a are arranged to form a droplet array. This operation is performed by repeating the operations (3) and (4) as many times as the number of droplets 41a arranged in the droplet array.
(6) The internal pressure of the pipette 14 is controlled by the pressure controller 12, and the reaction compound 42 (reagent 2) in the container 19a is sucked into the pipette 14 filled with oil.
(7) The internal pressure of the pipette 14 is controlled by the pressure controller 12, and the reaction compound 42 is discharged from the pipette 14 to form a droplet 42a of the reaction compound 42.
(8) The pipette 14 is moved by the XYZ stage 13 to bring the droplet 42 a of the reaction compound 42 into contact fusion with the droplet 41 a of the target compound 41 arranged on the bottom surface of the container 16. Here, the contact-fused droplet 41 a is indicated by a droplet 51.
(9) The presence or absence of reaction of the droplet 51 is analyzed by the observation system (observation system 20 and control system 11).
(10) Based on the result of the analysis, the reacted droplet 51 (compound) is used as a chemical candidate.
The operations (8) to (10) are performed as many times as the number of the reaction compounds 42, and all the reaction compounds 42 are examined for the presence of reaction. Then, the reaction compound 42 that has reacted is set as a drug candidate substance.

  As described above, the screening apparatus 10 of the present embodiment checks the presence or absence of reaction for a plurality of combinations of the reagent 1 and the reagent 2 by the procedures (1) to (10), and the reaction compound 42 ( Search for reagent 2).

[Automation of screening equipment]
In the screening apparatus 10 of the present embodiment, the search for drug candidate substances is automated. This automation is performed under the control of the control system 11.

FIG. 4 is a flowchart showing a control process of the entire system of the control system 11. The processing of the control system 11 will be described with reference to FIG.
The XYZ stage 13 is moved, and the pipette 14 is put into the container 16 filled with the oil 31 (S1). The pressure controller 12 is controlled to cause the pipette 14 to suck the oil 31 in the container 16 (S2). The first XY stage 15 carrying the reagent 1 library 18 is moved to the vicinity of the pipette 14 (S3). The XYZ stage 13 is moved, and the pipette 14 is put into the reagent 1 container 18a (S4). The pressure controller 12 is controlled to cause the pipette 14 to suck the reagent 1 in the container 18a (S5).

  The XYZ stage 13 is moved, and the tip of the pipette 14 is inserted to the vicinity of the bottom surface of the container 18a (S6). In this case, since the container 18 a is filled with the oil 31, the tip of the pipette 14 enters the oil 31.

  Next, the pressure controller 12 is controlled to discharge the reagent 1 from the tip of the pipette 14 (S7). In this case, a droplet of the reagent 1 is formed at the tip of the pipette 14 and the droplet is disposed on the bottom surface of the container 16.

Subsequently, it is determined whether or not a predetermined number of droplets (reagent 1 droplets) have been formed on the bottom surface of the container 16 (S8). If the number of droplets has not reached the predetermined number, the process returns to step S4.
In this step S4, the XYZ stage 13 is moved, and the pipette 14 is moved to the vicinity of the container 18a containing the reagent 1 to be aspirated next. Thereafter, the processes in steps S5 to S7 described above are performed.

  In this way, the processes in steps S4 to S7 are repeated until a predetermined number of droplets (reagent 1 droplets) are formed on the bottom surface of the container 16. Through the above process, a droplet array in which a predetermined number of droplets (reagent 1 droplets) are arranged is formed on the bottom surface of the container 16.

When it is determined in step S8 that a predetermined droplet (reagent 1 droplet) has been formed on the bottom surface of the container 16, the process proceeds to step S9.
Since the amount of the oil 31 sucked by the pipette 14 in step S1 is a sufficient amount, the oil 31 still remains in the pipette 14 at the time of shifting to step S9.

  In step S 9, the second XY stage 17 on which the reagent 19 library 19 is mounted is moved to the vicinity of the pipette 14. Next, the XYZ stage 13 is moved, and the pipette 14 is put into the container 19a in which the reagent 2 is stored (S10). The pressure controller 12 is controlled to cause the pipette 14 to aspirate the reagent 2 in the container 19a (S11).

  The XYZ stage 13 is moved, and the tip of the pipette 14 is put into the container 16 (S12). In this case, the tip of the pipette 14 is positioned in the vicinity of the droplet of the reagent 1 formed on the bottom surface of the container 16.

  The pressure controller 12 is controlled to discharge the reagent 2 from the tip of the pipette 14 and form a droplet of the reagent 2 on the tip of the pipette 14 (S13). The XYZ stage 13 is moved, and the droplet of the reagent 2 formed on the tip of the pipette 14 is brought into contact with the droplet of the reagent 1 disposed on the bottom surface of the container 16 (S14).

  The observation system 20 is photographed by the contact fusion result, and the photographed image is transmitted to the control system 11. The control system 11 determines the presence / absence of a reaction between the reagent 1 and the reagent 2 based on the received image (S15).

Next, it is determined whether or not the fusion (contact fusion) of the droplet of reagent 1 and the droplet of reagent 2 has been repeated a predetermined number of times (S16). If the predetermined number of times has not yet been reached, the process returns to step S10.
In the next step S10, the XYZ stage 13 is moved so that the pipette 14 enters the container 19a containing the reagent 2 to be next fused. Thereafter, the processes of steps S12 to S15 described above are performed.

  In this way, contact fusion of the droplet of reagent 1 and the droplet of reagent 2 is performed a predetermined number of times in a plurality of combinations, and the presence or absence of reaction in contact fusion by each combination is examined. If it is determined in step S16 that the number of times of contact fusion has reached a predetermined number, the processing of this flowchart is terminated.

  As described above, the screening apparatus 10 of the present embodiment performs the process of fusing each reaction compound 42 (reagent 2) of the library 18 of reagent 1 and each target compound 41 (reagent 1) of the library 19 of reagent 2; The process of investigating the presence or absence of a reaction due to the fusion and searching for the reagent 1 as a drug candidate substance is automated.

[Drop formation of reagent 1]
FIG. 5 is a diagram showing details of steps (3) to (5) in FIG. With reference to FIG. 5, a method for producing a droplet array of the reagent 1 on the bottom surface of the container 16 will be described. FIG. 5 shows the operation after the pipette 14 is filled with the oil 31. The pipette 14 is assumed to be attached to the XYZ stage 13 in the vertical direction.

(1) The pipette 14 filled with the oil 31 is moved to a position above the second XY stage 17 by the XYZ stage 13.
(2) The library 18 of the reagent 1 is moved to the vicinity of the pipette 14 by the second XY stage 17. At this time, the container 18 a containing the target reagent 1 is positioned below the pipette 14.
(3) The pipette 14 is lowered into the container 18a by the XYZ stage 13. Then, the pressure controller 12 causes the pipette 14 to aspirate the reagent 1 in the container 18a.
(4) The pipette 14 is moved above the container 16 by the XYZ stage 13.
(5) The pipette 14 is brought close to the bottom surface of the container 16 by the XYZ stage 13. Then, the reagent 1 is discharged from the pipette 14 by the pressure controller 12, and a droplet 41 a of the reagent 1 is formed at the tip of the pipette 14. Then, the droplets 41 a are arranged on the bottom surface of the container 16. The volume of the droplet 41a is 1 fl (femtoliter) to 1 nl (nanoliter).
(6) The above operations (3) to (5) are repeated a predetermined number of times to arrange the reagent 1 droplets 41a on the bottom surface of the container 16 to form a reagent 1 droplet array.

[Fusion of Reagent 1 and Reagent 2]
FIG. 6 is a diagram showing details of steps (6) to (8) in FIG. A method for bringing the reagent 1 and the reagent 2 into contact with each other in the container 16 will be described with reference to FIG.

(1) The pipette 14 containing oil 31 (not shown) is moved to a position above the second XY stage 17 by the XYZ stage 13.
(2) The library 19 of the reagent 2 is moved to the vicinity of the pipette 14 by the second XY stage 17. At this time, the container 19 a containing the target reagent 2 is positioned below the pipette 14.
(3) The pipette 14 is lowered into the container 19a by the XYZ stage 13. Then, the pressure controller 12 causes the pipette 14 to suck the reagent 2 in the container 19a.
(4) The pipette 14 is moved above the container 16 by the XYZ stage 13.
(5) The pipette 14 is brought close to the bottom surface of the container 16 by the XYZ stage 13. Then, the reagent 2 is discharged from the pipette 14 by the pressure controller 12, and a droplet of the reagent 2 is formed at the tip of the pipette 14. The volume of the droplet 42a is 1 fl (femtoliter) to 1 nl (nanoliter). Subsequently, the pipette 14 is lowered by the XYZ stage 13, and the droplet 42 a formed at the tip of the pipette 14 is brought into contact with the droplet 41 a of one reagent 1 disposed on the bottom surface of the container 16.
(6) The above operations (3) to (5) are repeated a predetermined number of times, and contact fusion of the droplet 41a of the reagent 1 and the droplet 42a of the reagent 2 is performed in a plurality of combinations.

[Formation and Arrangement of Droplet Array in Container 16]
Next, each example of the configuration of the reagent 1 droplet array formed on the bottom surface of the container 16 will be described.

{Example 1}
FIG. 7 is a diagram showing a first embodiment of the reagent 1 droplet array formed on the bottom surface of the container 16. 7A and 7B are a top view and a side view of the container 16, respectively.

  As shown in FIG. 7A, on the bottom surface 16a of the container 16, the droplets 41a of the reagent 1 are arranged in a lattice pattern at almost equal intervals. The material of the container 16 is, for example, plastic or glass, and is usually not completely hydrophobic. For this reason, the droplet 41a of the reagent 1 is fixed to the bottom surface 16a only by contacting the bottom surface 16a of the container 16. The thickness of the oil 31 layer filled in the container 16 is, for example, 500 μm to 5 mm.

{Example 2}
FIG. 8 is a view showing a second embodiment of the droplet array of the reagent 1 formed on the bottom surface of the container 16. 8A and 8B are a top view and a side view of the container 16, respectively. FIG. 8C is a diagram showing the shape of the reagent 1 droplet formed on the bottom surface of the container 16.

  In this embodiment, as shown in FIG. 8B, the bottom surface 16a of the container 16 is subjected to a hydrophilic treatment. Specifically, the entire bottom surface 16a of the container 16 is made of a material (coating material) having higher hydrophilicity than the oil 31. ) 61 to coat. In this way, by coating the entire bottom surface of the container 16 with the highly hydrophilic material 61, the droplet 41a of the reagent 1 can be stabilized on the bottom surface 16a of the container 16 even when the material of the container 16 is hydrophobic. Can be formed.

  FIG. 8C is an enlarged view of a portion surrounded by a broken line in FIG. 8B, and shows the shape of two adjacent droplets 41 a formed on the material 61. Since the droplet 41a is easily wetted by the material 61, it has a substantially hemispherical shape.

{Example 3}
FIG. 9 is a view showing a third embodiment of the droplet array of the reagent 1 formed on the bottom surface of the container 16. 9A and 9B are a top view and a side view of the container 16, respectively. FIG. 9C is a diagram showing the shape of the droplet of reagent 1 formed on the bottom surface of the container 16.

  In the present embodiment, as shown in FIG. 9A, the bottom surface 16a of the container 16 is partially hydrophilized. As shown in FIGS. 9A and 9C, this hydrophilization is performed by applying a hydrophilization treatment to the position where the droplet 41a of the reagent 1 is formed. As shown in FIG. 9A, the hydrophilic treatment is performed by arranging the hydrophilic regions 71 in a spot shape in advance at positions where the droplets 41a on the bottom surface 16a of the container 16 are to be formed. As shown in FIG. 9C, which is an enlarged view of the broken line part of FIGS. 9A and 9B, the shape of the hydrophilic region 71 is smaller than the cross section at the center of the sphere of the droplet 41a. Make a circle. Since the liquid droplet 41a is held on the hydrophilic region 71 by surface tension, the shape of the liquid droplet 41a is almost spherical as shown in FIG. 9C, where only the bottom surface in contact with the hydrophilic region 71 is flat. It becomes a shape close to.

  For example, hexamethyldisilazane is used as the coating material (the material used to form the raw material 61 and the hydrophilic region 71) used in Examples 2 and 3 above.

[Details of reagent aspiration method with pipette 14]
FIG. 10 is a diagram showing a method of aspirating the reagent (reagent 1, reagent 2) with the pipette 14. FIG. 10 shows a method for aspirating the reagent 1. Reagent 2 is also aspirated in the same manner as reagent 1.

(1) As shown in FIG. 10A, the pipette 14 filled with the oil 31 is moved above the container 18a containing the reagent 1. In the container 18 a, a reagent 1 layer and an oil 31 layer are formed. The lower layer is the reagent 1 layer and the upper layer is the oil 31 layer. The oil 31 layer is thinner than the reagent 1 layer.
(2) As shown in FIG. 10 (b), the pipette 14 is lowered into the container 18 a to a position where the tip portion reaches the inside of the reagent 1 layer. Then, the reagent 1 is aspirated into the pipette 14.
(3) As shown in FIG. 10C, the pipette 14 is pulled up to the vicinity of the upper part of the oil 31 layer. In this process, the oil 31 adheres to the tip of the pipette 14. This is because the oil 31 has adhesiveness.
(4) The pipette 14 is further pulled up, and its tip is pulled away from the oil 31 layer. At this time, the tip portion of the pipette 14 is blocked by the attached oil 31. As described above, the tip portion of the pipette 14 is blocked by the oil 31, so that the volatilization rate of the reagent 1 is reduced while the reagent 1 is transported to the upper side of the container 16 by the pipette 14.

[Reaction Compound Supply Method]
Next, a method of bringing the reagent 2 as the reaction compound 42 into contact fusion with the reagent 1 as the target compound 41 will be described with reference to FIG.

  First, as shown in FIG. 11A, the tip of the pipette 14 is brought close to the vicinity of the reagent 41 droplet 41 a formed on the bottom surface 16 a of the container 16 filled with the oil 31. Then, the reagent 2 is discharged from the tip of the pipette 14 at that position, and a droplet 42 a of the reagent 2 is formed on the tip of the pipette 14.

  Next, as shown in FIG. 11 (b), the pipette 14 is brought closer to the droplet 41a of the reagent 1, and the droplet 2a of the reagent 2 formed at the tip of the pipette 14 is brought into contact with the droplet 41a, so that the liquid The droplet 42a is fused to the droplet 41a. As a result, as shown in FIG. 11C, the droplet 42a diffuses into the droplet 41a, and when the reagent 2 is the target compound, the droplet 42a and the droplet 41a react. The reaction between the reagent 1 droplet 41a and the reagent 2 droplet 42a can be detected by, for example, a quenching phenomenon of fluorescence intensity. In this case, for example, the phenomenon of fluorescence intensity decreases due to quenching. This phenomenon is captured by the camera of the observation system 20, and the captured image is transmitted from the observation system 20 to the control system 11. The observation system 20 performs image processing on the received photographed image, and detects whether or not there is a reaction between the reagent 41 droplet 41a and the reagent 2 droplet 42a.

  By the way, a hydrophobic coating may be applied to the inside of the pipette 14. In this case, for example, hexamethyldilasan is used as the coating material. Since the pipette 14 is a glass material and the glass is hydrophilic, when the reagent 1 or the reagent 2 is discharged from the pipette 14, these reagents may adhere to the pipette and it may be difficult to form droplets. Therefore, by applying a hydrophobic coating to the inside of the pipette 14, it is possible to smoothly form droplets (reagent 1 droplet 41 a, reagent 2 droplet 42 a).

[Configuration of Container 16 Forming Droplet Array]
Since the oil has a hygroscopicity of ppm level, the reagent 1 droplet 41a disposed in the oil 31 shrinks at a speed of 10 to 100 fl / s under a normal temperature and normal humidity environment. For this reason, unless any countermeasure is taken, the droplet 41a of the reagent 1 in the droplet array formed in the container 16 shrinks with time.
Two countermeasures are proposed for this purpose.

{Example 1}
In the first embodiment, a groove is provided around the reagent 1 droplet array, and water is retained in the groove. By adopting such a configuration, absorption of water into the oil 31 is performed not only from the droplet 41a of the reagent 1 but also from surrounding water. As a result, the water absorption rate from the droplet 41a of the reagent 1 by the oil 31 is relaxed. In this case, water may be injected into the groove in advance, and then the oil 31 may be injected into the container 16, or the injection order may be reversed.
FIG. 12 is a diagram showing a first example of the configuration of the container 16 for relaxing the water absorption of the droplet 41a of the reagent 1 by the oil 31. FIG. 12A and 12B are a top view and a side view of the container 16, respectively.

  In this embodiment, as shown in FIGS. 12A and 12B, a substantially rectangular groove 101 is provided around the reagent 1 droplet array. In this configuration, the periphery of the oil 31 is surrounded by the water 103 contained in the groove 101, and the oil 31 absorbs moisture from the water 103 inside the groove 101.

{Example 2}
In the second embodiment, a droplet 105 of the solvent of the reagent 1 is arranged around the droplet array of the reagent 1.

  FIG. 13 is a diagram showing a second example of the configuration of the container 16 that relaxes the water absorption of the droplets 41 a of the reagent 1 by the oil 31. 13A and 13B are a top view and a side view of the container 16, respectively.

  In this embodiment, as shown in FIGS. 13A and 13B, the solvent droplet 105 of the reagent 1 (hereinafter simply referred to as the droplet 105 for the sake of convenience) around the droplet 41a of the reagent 1. Form). In the case of this example, a plurality of droplets 105 are arranged around the droplet array at the same interval as the arrangement pitch of the droplets 41a so as to surround the droplet array of the droplets 41a. For example, the droplet 105 is formed by a method similar to the method of forming the droplets of the reagent 1 and the reagent 2 described above. In the case of the present embodiment, the oil 31 absorbs moisture from the droplet 105, so the rate of decrease in the moisture concentration of the droplet 41a of the reagent 1 is alleviated.

By the way, in the present embodiment, the oil 31 is used as the liquid for arranging the droplet array of the reagent 1, but the present invention is not limited to this. It is also possible to use liquids other than oils that are not compatible (melted with each other) with the reagent solution.
Further, the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the spirit of the present invention.
(Appendix 1)
A droplet array means in which a droplet array of the first reagent is formed in a liquid incompatible with the first reagent;
Fusing means for contacting and fusing a second reagent drop to a first reagent drop disposed on the drop array means;
A screening apparatus comprising:
(Appendix 2)
The first reagent is ejected in a liquid that is incompatible with the first reagent to form the first droplet, and the droplet is arranged on the droplet array means to form the first reagent. A droplet array forming means for forming a droplet array of
The screening apparatus according to appendix 1, further comprising:
(Appendix 3)
The droplet array forming means includes:
A suction means for sucking the first reagent after being filled with a liquid that is incompatible with the first reagent;
The first reagent is ejected from the suction means in a liquid that is incompatible with the first reagent to form a droplet of the first reagent, and the droplet is disposed on the droplet array means. A droplet formation control means for
The screening apparatus according to appendix 2, which is provided.
(Appendix 4)
The suction means is a pipette;
The droplet formation control means controls the internal pressure of the pipette to cause the pipette to suck the liquid that is incompatible with the first reagent and the first reagent, and to discharge the first reagent from the pipette. The screening apparatus according to appendix 3, wherein a droplet of the first reagent is formed.
(Appendix 5)
A first storage means for storing a layer of the first reagent and a layer of liquid that is incompatible with the first reagent provided on the layer;
The screening apparatus according to appendix 3, wherein the aspirating unit aspirates the first reagent from the first accommodating unit.
(Appendix 6)
The liquid that is incompatible with the first reagent is a material that adheres to a tip portion of the suction means when the suction means sucks the first reagent from the first storage means. The screening apparatus according to appendix 5.
(Appendix 7)
The fusion means includes
A suction means for sucking the second reagent after being filled with a liquid that is incompatible with the first reagent;
Fusion control means for discharging the second reagent from the suction means and bringing the second reagent droplet into contact fusion with the droplet of the first reagent arranged on the droplet array means ,
The screening apparatus according to appendix 1, wherein the screening apparatus is provided.
(Appendix 8)
A second storage means for storing a layer of the second reagent and a layer of liquid that is incompatible with the second reagent provided on the layer;
The screening apparatus according to appendix 7, wherein the aspirating unit aspirates the second reagent from the second storage unit.
(Appendix 9)
The liquid that is incompatible with the second reagent is a material that adheres to the tip of the suction means when the suction means sucks the second reagent from the second storage means. The screening apparatus according to appendix 8.
(Appendix 10)
The screening apparatus according to appendix 8 or 9, wherein the liquid not compatible with the first reagent and the liquid not compatible with the second reagent are the same liquid.
(Appendix 11)
The droplet array means comprises a replenishing means for replenishing a liquid that is incompatible with the first reagent with a liquid that is incompatible with the first reagent, a component that is absorbed from the liquid droplets of the first reagent.
The screening apparatus according to appendix 1, wherein the screening apparatus is provided.
(Appendix 12)
The replenishing means is
12. The screening apparatus according to appendix 11, wherein the screening apparatus is provided around a droplet array of the first reagent arranged on the droplet array means.
(Appendix 13)
The replenishing means is
13. The screening apparatus according to appendix 12, wherein a groove provided around the first reagent droplet array is configured to contain the solvent of the first reagent.
(Appendix 14)
The replenishing means is
13. The screening apparatus according to appendix 12, wherein the first reagent droplets are arranged so as to surround the first reagent droplet array.
(Appendix 15)
The screening apparatus according to appendix 1, wherein the liquid that is incompatible with the first reagent is used in a state in which absorption of components from the first reagent is in equilibrium.
(Appendix 16)
The screening apparatus according to appendix 1, wherein the liquid incompatible with the first reagent is oil.
(Appendix 17)
A liquid forming liquid that is incompatible with the reagent and a liquid droplet forming means that forms a liquid droplet of the moving reagent by sucking the liquid and discharging the liquid
Pressure control means for controlling the internal pressure of the droplet forming means to cause the droplet forming means to perform a liquid incompatible with the reagent, an operation of sucking the reagent, and an operation of discharging the moving reagent;
First storage means for storing a liquid that is incompatible with the reagent;
A second containing means for containing the first reagent;
A third storage means for storing the second reagent;
The droplet forming means is moved and the pressure applied by the pressure control means to the droplet forming means is controlled to be disposed in a liquid incompatible with the first reagent in the first storage means. Forming a droplet array of one reagent, and causing the droplet forming means to form droplets of the second reagent, and causing the second reagent droplets to form the second reagent in the droplet array. A control means for contact fusion with each droplet of
A screening apparatus comprising:
(Appendix 18)
The screening apparatus according to appendix 1 or 17, wherein the first reagent is a target compound, and the second reagent is a candidate compound for examining the presence or absence of a reaction with the target compound.
(Appendix 19)
A contact fusion step of contacting and fusing a second reagent droplet to a first reagent droplet formed in a liquid that is incompatible with the first reagent;
A screening method characterized by comprising.
(Appendix 20)
The first reagent is ejected in a liquid that is incompatible with the first reagent to form the first droplet, and the droplet is arranged on the droplet array means to form the first reagent. A droplet array forming step to form a droplet array of
The screening method according to claim 19, further comprising:
(Appendix 21)
The droplet array forming step includes
After the suction means is filled with a liquid incompatible with the first reagent, the suction means sucks the first reagent; and
The first reagent is ejected from the suction means in a liquid that is incompatible with the first reagent to form droplets of the first reagent, and the droplets are arranged to form the droplet array. Step to
The screening method according to appendix 20, wherein the screening method is provided.
(Appendix 22)
The suction means is a pipette;
By controlling the internal pressure of the pipette, the pipette sucks the liquid that is incompatible with the first reagent and the first reagent, and discharges the first reagent from the pipette. The screening method according to appendix 21, wherein the liquid droplets are formed.
(Appendix 23)
The suction means sucks the first reagent from the first storage means for storing the first reagent layer and the liquid layer incompatible with the first reagent provided on the first reagent layer. The screening method according to appendix 21, wherein:
(Appendix 24)
The liquid that is incompatible with the first reagent is a material that adheres to a tip portion of the suction means when the suction means sucks the first reagent from the first storage means. The screening method according to appendix 23.
(Appendix 25)
The contact fusion step includes
Filling the suction means with a liquid that is incompatible with the first reagent, and then causing the suction means to suck the second reagent;
A fusion control step of discharging the second reagent from the suction means and bringing the second reagent droplet into contact fusion with the first reagent droplet disposed in the droplet array;
The screening method according to appendix 19, characterized by comprising:
(Appendix 26)
The second reagent is aspirated from the second accommodating means for accommodating the second reagent layer and the liquid layer incompatible with the second reagent provided on the second reagent layer. The screening method according to appendix 25, wherein
(Appendix 27)
The liquid that is incompatible with the second reagent is a material that adheres to the tip of the suction means when the suction means sucks the second reagent from the second storage means. The screening method according to appendix 26.
(Appendix 28)
28. The screening method according to appendix 26 or 27, wherein the liquid incompatible with the first reagent and the liquid incompatible with the second reagent are the same liquid.
(Appendix 29)
The screening method according to appendix 19, wherein a liquid that is incompatible with the first reagent is supplemented with a component that the liquid absorbs from the droplets of the first reagent in the droplet array.
(Appendix 30)
30. The screening method according to appendix 29, wherein the absorbing component is replenished from around the droplet array of the first reagent.
(Appendix 31)
31. The screening method according to appendix 30, wherein the absorption component is replenished from a solvent of the first reagent housed in a groove provided around the droplet array of the first reagent.
(Appendix 32)
31. The screening method according to appendix 30, wherein the absorbing component is replenished from droplets of the solvent of the first reagent arranged so as to surround the droplet array of the first reagent. (Appendix 33)
The screening method according to appendix 20, wherein the liquid incompatible with the first reagent is used in a state where the absorption of components from the first reagent is in equilibrium.
(Appendix 34)
The screening method according to appendix 20, wherein the liquid incompatible with the first reagent is oil.
(Appendix 35)
A liquid droplet forming step of forming a liquid droplet of the reagent by causing the liquid droplet forming means to suck the liquid that is incompatible with the reagent and discharging the reagent from the liquid droplet forming means;
A pressure control step of controlling the internal pressure of the droplet forming means to cause the droplet forming means to perform a liquid incompatible with the reagent, an operation of sucking the reagent, and an operation of discharging the moving reagent;
The movement of the droplet forming means and the pressure applied by the pressure control means to the droplet forming means are controlled so as to be compatible with the first reagent in the first containing means for containing the liquid incompatible with the reagent A droplet array of a first reagent disposed in a liquid that is not to be formed, and a droplet of a second reagent to be formed in the droplet forming means, and the droplet of the second reagent is formed into the droplet array A control step of contacting and fusing each droplet of the first reagent therein;
A screening method comprising:
(Appendix 36)
36. The screening method according to appendix 19 or 35, wherein the first reagent is a target compound, and the second reagent is a candidate compound for examining the presence or absence of a reaction with the target compound.

It is a figure which shows the system configuration | structure of one Embodiment of the screening apparatus of this invention. It is a figure which shows the signal waveform for the internal pressure control of the pipette which a control system applies to a pressure controller. It is a figure which shows the basic procedure of the screening method using the screening apparatus 10 of this embodiment. It is a flowchart which shows the control processing of the whole system of a control system. It is a figure which shows the detail of process (3)-(5) of FIG. It is a figure which shows the detail of process (6)-(8) of FIG. It is a figure which shows the 1st Example of the droplet array of the reagent 1 formed in the bottom face of a container, (a), (b) is the top view and side view of a container, respectively. It is a figure which shows the 2nd Example of the droplet array of the reagent 1 formed in the bottom face of a container, (a), (b) is a top view and a side view of a container, respectively, (c) is a bottom face of a container. It is a figure which shows the shape of the droplet of the reagent 1 formed in FIG. It is a figure which shows the 3rd Example of the droplet array of the reagent 1 formed in the bottom face of the container 16, (a), (b) is the top view and side view of a container, respectively, (c) is the container 16 It is a figure which shows the shape of the droplet of the reagent 1 formed in the bottom face. It is a figure which shows the suction method of the reagent (reagent 1, reagent 2) with a pipette. It is a figure which shows the method of contact-fusing the reagent 2 which is a reaction compound with the reagent 1 which is a target compound. It is a figure which shows the 1st Example of the structure of the container which eases the water | moisture-content absorption of the droplet of the reagent 1 by oil, (a), (b) is the top view and side view of a container, respectively. It is a figure which shows the 2nd Example of the structure of the container which eases the water | moisture-content absorption of the droplet 41a of the reagent 1 by oil, (a), (b) is the top view and side view of a container, respectively.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Screening apparatus 11 Control system 11a Display 12 Pressure controller 13 XYZ stage 14 Pipette 15 First XY stage 16 Container 16a Bottom surface 17 Second XY stage 18 Reagent 1 library 18a Container (Reagent 1 container)
19 Reagent 2 library 19a Container (Reagent 2 container)
20 Observation System 31 Oil 41 Target Compound (Reagent 1)
41a droplet (target compound droplet)
42 Reaction Compound (Reagent 2)
42a droplet (reaction compound droplet)
51 droplets (droplets in which the droplets of reagent 1 and reagent 2 are fused in contact)
61 Highly hydrophilic material 71 Hydrophilic region 101 Groove (groove provided in the container 16)
103 water

Claims (10)

A droplet array means in which a droplet array of the first reagent is formed in a liquid incompatible with the first reagent;
Fusing means for contacting and fusing a second reagent drop to a first reagent drop disposed on the drop array means;
A screening apparatus comprising: The first reagent is ejected in a liquid that is incompatible with the first reagent to form the first droplet, and the droplet is arranged on the droplet array means to form the first reagent. A droplet array forming means for forming a droplet array of
The screening apparatus according to claim 1, further comprising: The droplet array forming means includes:
A suction means for sucking the first reagent after being filled with a liquid that is incompatible with the first reagent;
The first reagent is ejected from the suction means in a liquid that is incompatible with the first reagent to form a droplet of the first reagent, and the droplet is disposed on the droplet array means. A droplet formation control means for
The screening apparatus according to claim 2, further comprising: The suction means is a pipette;
The droplet formation control means controls the internal pressure of the pipette to cause the pipette to suck the liquid that is incompatible with the first reagent and the first reagent, and to discharge the first reagent from the pipette. 4. The screening apparatus according to claim 3, wherein a droplet of the first reagent is formed. A first storage means for storing a layer of the first reagent and a layer of liquid that is incompatible with the first reagent provided on the layer;
The screening apparatus according to appendix 3, wherein the aspirating unit aspirates the first reagent from the first accommodating unit.   The liquid that is incompatible with the first reagent is a material that adheres to a tip portion of the suction means when the suction means sucks the first reagent from the first storage means. The screening apparatus according to claim 5. The fusion means includes
A suction means for sucking the second reagent after being filled with a liquid that is incompatible with the first reagent;
Fusion control means for discharging the second reagent from the suction means and bringing the second reagent droplet into contact fusion with the droplet of the first reagent arranged on the droplet array means ,
The screening apparatus according to claim 1, further comprising: A second storage means for storing a layer of the second reagent and a layer of liquid that is incompatible with the second reagent provided on the layer;
The screening apparatus according to claim 7, wherein the suction unit sucks the second reagent from the second storage unit. The liquid that is incompatible with the second reagent is a material that adheres to the tip of the suction means when the suction means sucks the second reagent from the second storage means. The screening apparatus according to claim 8. The droplet array means comprises a replenishing means for replenishing a liquid that is incompatible with the first reagent with a liquid that is incompatible with the first reagent, a component that is absorbed from the liquid droplets of the first reagent.
The screening apparatus according to claim 1, further comprising: