JP2003106950A - Method for injecting microdroplets or microbubbles - Google Patents

Abstract

(57) [Problem] To provide a method for smoothly injecting minute droplets and minute bubbles ejected from a minute nozzle into a minute container. SOLUTION: A microdroplet or a microbubble ejected from a nozzle into a medium is captured at a focal position or a scanning center of a laser beam, and the focal position or the scanning center is moved, or on a scanning orbit of the laser beam or By capturing in the scanning cavity and moving the scanning trajectory or scanning cavity, it is guided into the microcontainer to inject microdroplets or microbubbles.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a femtoliter to picoliter amount of liquid ejected from a minute nozzle,
The present invention relates to a method for injecting gas or micrometer-sized fine particles into a micro container.

[0002]

2. Description of the Related Art Recently, in the fields of medical treatment, chemical analysis, environmental measurement, etc., a technology for micronizing members of a mixing / reaction system, a detection system, an electronic circuit, an actuating pump, etc., and making the entire system IC The development of so-called μ-TAS has become an important issue. As one of them, it is possible to drop a small amount of a test reagent into a micro container arranged in an array by using a minute nozzle. However, with this method, it has been difficult to inject a plurality of liquid reagents into the same array in an accurate amount or to inject a solid or gaseous reagent.

On the other hand, there is known a method for manipulating micro-sized fine particles or liquid droplets by using optical tweezers or laser scanning manipulation, and using this method, it is possible to carry out reaction in a minute region in the fields of chemistry and biotechnology. Manipulation of reagents and genes during analysis and analysis is being considered.

However, in this method, a large amount of droplets and fine particles to be manipulated must be prepared, and some of them must be observed and observed under a microscope, so most of the droplets and fine particles are not used. However, there is a problem that this causes disturbances when collecting laser light and obstruction of the path during movement operation, and these solutions are important for smoothly injecting only the required amount of reagent into a predetermined micro container. It was a challenge.

[0005]

SUMMARY OF THE INVENTION The present invention has been made for the purpose of providing a method for smoothly injecting fine droplets or fine bubbles ejected from a fine nozzle into a fine container.

[0006]

DISCLOSURE OF THE INVENTION The inventors of the present invention have made various studies on a method of injecting microdroplets or microbubbles ejected from a micronozzle into a microcontainer, which has been very difficult by the conventional techniques. As a result, by using laser manipulation technology, it is possible to easily inject microdroplets or microbubbles generated by ejecting a liquid or gas of femtoliter to picoliter from a micro nozzle into a micro container. The present invention has been completed based on this finding.

That is, the present invention captures minute droplets or minute bubbles ejected from a nozzle into a medium at the focal position or scanning center of laser light, and moves the focal position or scanning center to move the minute container. A method for injecting microdroplets or microbubbles, characterized in that they are guided inside, and trapping microdroplets or microbubbles ejected from a nozzle into a medium on a scanning trajectory of a laser beam or in a scanning cavity, The present invention provides a method for injecting microdroplets or microbubbles, which is characterized in that it is guided into a microcontainer by moving a scanning trajectory or a scanning cavity.

[0008]

The method of the present invention will be described below with reference to the accompanying drawings. FIG. 1 shows a state in which a microdroplet 3 such as a microfluidic paraffin droplet ejected into a medium 1 such as water from a micronozzle 2 such as a micropipette is captured and moved at a focus position (+) of laser light. It is a micrograph. In this figure, a very small amount of liquid paraffin injected from the micropipette 2 into the water 1 at an injection pressure Pi and an injection time Ti forms an oil droplet 3 having a diameter of about 3 μm [FIG. 1 (a)], and Pi Is applied for Ti seconds and the next oil droplet 3'is ejected, the first oil droplet 3 is captured at the focal position of the laser beam at the cross position in FIG. 1 (a) [FIG. 1 (b)]. The oil droplet 3 thus captured is moved to the lower left position [Fig. 1 (c)], and Pi
Is added for Ti seconds to eject the oil droplet 3 ″, the oil droplet 3 ′ is moved to the side adjacent to the oil droplet 3 by the second laser beam [FIG.
(D)]. Therefore, by arranging the micro-container at the lower left position, the oil droplets 3, 3 ', 3 "... Can be sequentially injected into this.

Next, FIG. 2 is an explanatory view in the case of ejecting minute droplets 3 and 3'of different kinds from two minute nozzles 2 and 2'and injecting them into minute containers 7 and 7 '. The droplet 3 from the minute nozzle 2 and the droplet 3'from the minute nozzle 2'are the medium 1
The transparent substrate 6 filled with the above is emitted to the upper portions of the micro-containers 7 and 7 ′ produced by processing, and captured by the laser beams A and B, respectively. The droplets 3 and 3 ′ captured here are three-dimensionally moved by moving the focal positions of the laser beams A and B condensed by the lens 8 from the vicinity of the nozzle to the bottom position of the minute container. Can be injected into the micro-containers 7, 7 '.

The ejection of a droplet from such a minute nozzle may not be reliably performed because the droplet after ejection adheres to the tip of the nozzle due to the relationship between the nature of the ejected droplet and the nozzle material and shape. In such a case, in such a case, ultrasonic waves are radiated to the solution near the tip of the minute nozzle by the ultrasonic wave generating elements 5 and 5'formed or attached to the minute water tank wall surfaces 4 and 4 '. The droplet can be dissociated from the nozzle and ejected. Further, the diameter and the number of the ejected droplets are changed by changing the ejection pressure, the ejection time, and the sustaining pressure.

In the method of the present invention, fine particle-containing droplets may be ejected from the fine nozzles 2 and 2'instead of the droplets. In this case, by making the type of the droplet the same as that of the medium, the droplet is separated from the minute nozzle, and at the same time, the droplet portion disappears, and only the contained fine particles are released into the medium.

Further, if the bottom surface of the micro-container is treated to be hydrophilic or hydrophobic depending on the type of the liquid droplets to be injected, it is possible to fix it after the injection. The immobilization in the case of fine particles can be performed by injecting ultraviolet curable droplets and fine particles into the same micro container and then irradiating with ultraviolet light.

When capturing and injecting liquid droplets or fine particles having a refractive index lower than that of the medium 1, a laser scanning manipulation method of scanning around the liquid droplets is used.

Next, FIG. 3 is a system diagram for explaining an example of a system configuration for three-dimensionally moving the focal positions of two laser beams from the vicinity of the minute nozzle to inside the minute container. In this figure, the laser light from the light source 10 is
The diameter is converted by the beam expander 11, and the half-wave plate 12
Through the first polarization beam splitters 13 and 13 ', and after passing through the electromagnetic shutters 14 and 14', respectively, the focus position coordinate XYZ under the microscope is controlled by the computer Cp so as to be a desired position. The path is controlled by the focus position moving mechanisms 15 and 15 ′ which are composed of a galvano mirror pair and a focus position changing lens.

The above-mentioned decomposed laser light is again made coaxial by the second polarization beam splitters 16 and 16 ', and then introduced into the microscope 18 by being made coincident with the axis of the epi-illumination light by the relay lens 17. Is collected by a condenser lens 8 to a spot size of about 1 μm, for example, so as to be in the vicinity of the minute nozzles, and is irradiated to capture the ejected droplets. After that, as already described in FIG. 2, the focus position moving mechanisms 15 and 15 ′ three-dimensionally move the focus position to the desired position of the micro-container 7 to accurately inject the droplets. You can

At this time, since the polarization directions of the two laser beams are orthogonal to each other, interference does not occur. Therefore, since the intensity distribution does not change depending on the relative position of the two beams, each laser beam is different. Target droplets can be captured and injected in a stable state. In addition, the movement path, movement speed, scanning position, scanning speed, synchronous scanning phase difference, and scanning pattern of the focal points of the two laser beams are the CCD camera 19 indicating the droplet ejection state, the laser beam capture state, and the transportation state. Observed at
By processing the image with the image processing apparatus 20, it is possible to automatically change the image according to the purpose.

Thus, in the method of the present invention,
As shown in FIG. 1, not only the minute liquid droplets sequentially ejected from one minute nozzle, but also the minute liquid droplets ejected one by one from a plurality of minute nozzles as shown in FIG. It is possible to accurately inject a plurality of fine droplets sequentially ejected from each of the minute nozzles into the minute container.

During these injections, the injection pressure, the injection time, and the maintenance pressure are selected while observing the state of the microdroplets ejected from the micronozzles by image processing or by the data measured in advance. The diameter and number of microdroplets can be controlled by changing at least one factor that is set.

The method of the present invention has been described above with reference to an example in which minute droplets or minute droplets containing fine particles are ejected into the liquid medium and the focal position of the laser beam is used. However, they are ejected from the minute nozzles into the liquid medium. When using the movement of the micro bubbles and the scanning center, the scanning trajectory, or the scanning cavity, it is possible to inject into a predetermined micro container in the same manner. In this case, it is advantageous to collect the microbubbles with the opening of the container facing downward and the bottom facing upward.

[0020]

EXAMPLES Next, the present invention will be described in more detail by way of examples.

Example 1 In the system shown in FIG. 2 or 3, continuous wave Nd: YAG laser (wavelength 1
064 nm, linearly polarized light) was used to fill a distilled water (refractive index 1.33) in a micro water tank having a micro container on the bottom, which was created by etching a microscope cover glass.
By irradiating two laser beams from below through an oil immersion objective lens (magnification of 100) of a microscope, minute liquid droplets (liquid paraffin, refractive index 1.46) ejected from a minute nozzle formed by processing a glass capillary. ) And injection into a micro container. That is, the injection pressure, the injection time, and the maintenance pressure are controlled by a pump from a minute nozzle, and the size is 3 μm.
FIG. 1 shows the result of observing with a microscope how two droplets of each of the above are ejected in total, each of which is captured by the optical tweezers method using two laser beams, moved, and injected into a micro container.

FIG. 1A shows the injection pressure (30
(00 hPa) is applied for a set ejection time (0.1 seconds), and one droplet having a size of 3 μm is formed at the tip of the nozzle. The white cross in the figure shows the focal position of the laser light set near the tip of the nozzle in real time after image processing. FIG. 1B shows the second injection pressure (3000
hPa) is added for 0.1 second, and a state in which the droplet at the nozzle tip is ejected and captured at the focus position of the laser beam at the white cross position is shown. At this time, the next droplet is prepared at the tip of the nozzle. FIG. 1C shows a state in which the captured droplet is moved to the position of the micro container by the computer control of the focus position moving mechanism 15 of FIG. FIG. 1D shows the third injection pressure (3000
hPa) is applied for 0.1 second to eject the second droplet, and then the droplet is captured by the second beam, and is aligned in the X direction at the position next to the droplet in FIG. 1C. Is shown.

Example 2 Using the same system as in Example 1, injection pressure, injection time,
By changing the maintenance pressure, a total of two droplets having different diameters were ejected, and each droplet was captured and moved by an optical tweezer method using a laser beam to be injected into a micro container. FIG. 4 shows a state in which the ejected large and small droplets are captured by two beams and then arranged in the Y direction on the micro container. From this, it is understood that by controlling the ejection pressure, the ejection time, and the sustaining pressure, it is possible to selectively eject droplets of different sizes and move and inject them precisely.

The present invention is not limited to the above embodiments, and the design can be changed as appropriate, such as the optical system for introducing laser light, the positions, shapes and numbers of the minute nozzles and the ultrasonic wave generating elements.

[0025]

EFFECTS OF THE INVENTION According to the present invention, it is possible to automatically inject and immobilize a very small amount of liquid, gas and particulate reagents for constructing μ-TAS into a micron-sized array container. Not only that, it is possible to construct a sensitive μ-TAS or micrometer-order chemical reaction field and microbial growth field without the occurrence of disturbance fields and contamination due to excess reagents, thereby controlling the reaction and growth. It is also possible to do.

[Brief description of drawings]

FIG. 1 is a micrograph showing a process of carrying and injecting liquid droplets ejected from a minute nozzle according to the present invention.

FIG. 2 is an explanatory diagram showing an example of an injection method of the present invention.

FIG. 3 is a system diagram showing an example of the configuration of the present invention.

FIG. 4 is a micrograph showing a state in which droplets having different diameters are ejected and moved and injected.

[Explanation of symbols]

1 medium 2,2 'micro nozzle 3,3 'microdroplets 4,4 'Micro water tank wall surface 5,5 'Ultrasonic generator 6 Transparent substrate material 7,7 'micro container 8 Condensing lens 9 pumps 10 Laser light source 11 beam expander 12 Half-wave plate 13,13 'first polarization beam splitter 14,14 'Electromagnetic shutter 15,15 'Focus position moving mechanism 16,16 'second polarization beam splitter 17 relay lens 18 microscope 19 CCD camera 20 Image processing device

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Yosuke Kiuchi             3-3-14-2 Minamiyasancho, Tokushima City, Tokushima Prefecture (72) Inventor Satoshi Fukuoka             2217-14 Hayashi-cho, Takamatsu-shi, Kagawa Independent administrative agency             AIST Shikoku Center F term (reference) 2G052 AD26 AD42 AD46 CA04 CA07                       CA18 DA09                 2G058 AA03 AA09 EA11 EA14 ED11                 5F072 JJ20 MM14 MM17 YY01 YY11

Claims (8)

[Claims] 1. A microdroplet or a microbubble ejected from a nozzle into a medium is captured at a focal position or scanning center of laser light, and the focal position or scanning center is moved,
A method of injecting microdroplets or microbubbles, characterized by inducing into a microcontainer. 2. A microdroplet or a microbubble ejected from a nozzle into a medium is trapped on a scanning orbit of a laser beam or in a scanning cavity, and the scanning orbit or scanning cavity is moved so that the microcontainer is placed in a microcontainer. A method of injecting microdroplets or microbubbles, characterized by inducing. 3. The injection method according to claim 1, wherein the fine droplets or the fine bubbles are fine droplets or fine bubbles ejected one by one from a plurality of fine nozzles. 4. The injection method according to claim 1, wherein the fine droplets or the fine bubbles are fine droplets or fine bubbles sequentially ejected from one fine nozzle. 5. The injection method according to claim 1, wherein the fine droplets or the fine bubbles are a plurality of fine droplets or fine bubbles ejected in sequence from a plurality of fine nozzles per nozzle. 6. At least one selected from an injection pressure, an injection time, and a sustaining pressure while observing the state of micro droplets or micro bubbles ejected from micro nozzles by image processing.
The injection method according to any one of claims 1 to 5, wherein the diameter and the number of microdroplets or microbubbles are controlled by changing two factors. 7. The injection method according to claim 1, wherein the injection is performed while applying an ultrasonic wave near the tip of the minute nozzle. 8. The injection method according to claim 1, wherein fine particles are contained in the fine droplets.