JP3837484B2 - Trace sampling method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method in which a liquid or gas sample is ejected from a micro nozzle as micro droplets or micro bubbles of femtoliters to picoliters and collected in a micro container.
[0002]
[Prior art]
Recently, in various fields such as medical treatment, chemical analysis, environmental measurement, etc., a technology that makes the entire system into an IC by micro-mixing components such as mixing / reaction system, detection system, electronic circuit, working pump, so-called μ-TAS Development of has become an important issue, and as one of them, it is carried out by using a micro nozzle to drop a micro droplet of a test reagent into a micro container arranged in an array, or taken same Areihe plurality of liquid reagent in the correct amounts in this way, it is difficult to collect solid or gaseous reagents.
[0003]
On the other hand, a method of manipulating micro-sized particles and droplets using optical tweezers or laser scanning manipulation is known, and this method is used to analyze and analyze reactions in the micro area in the chemistry and biotechnology fields. In this case, it has been studied to manipulate reagents and genes.
[0004]
However, in this method, since a large amount of droplets and fine particles to be manipulated are prepared and operated while observing a part of them under a microscope, most of the droplets and fine particles remain unused, This has the problem of causing disturbances during laser beam condensing and path failures during moving operations, and these solutions are important issues in smoothly collecting only the necessary amount of reagent in a predetermined micro container . It was.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for ejecting a liquid or gas sample from a micro nozzle as a micro droplet or micro bubble into a liquid medium that is immiscible with the sample and smoothly collecting the liquid or gas sample in a micro container. It was made.
[0006]
[Means for Solving the Problems]
The present inventors have conducted various studies on a method of collecting a minute amount of liquid or gas sample in a micro container, which has been considered very difficult by the prior art. As a result, if laser manipulation technology is used, femtoliter It was found that microdroplets or microbubbles generated by injecting a picoliter amount of liquid or gas from a micronozzle into a liquid medium can be easily collected in a microcontainer. It came to an eggplant.
[0007]
That is, the present invention, when collecting a trace amount of liquid or gas sample in a micro container, ejects the sample as a micro droplet or micro bubble from a nozzle into a liquid medium immiscible with the sample. A method for collecting a small amount of a sample into a micro container, wherein the bubble is captured at the focal position or scanning center of the laser beam, and the focal position or scanning center is moved to be guided and accommodated in the micro container. And the micro droplet or micro bubble is captured on the scanning trajectory of the laser beam or in the scanning cavity, and is guided and accommodated in the micro container by moving the scanning trajectory or the scanning cavity. A method for collecting a small amount of sample into a micro container is provided.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Next, the method of the present invention will be described with reference to the accompanying drawings.
FIG. 1 shows a liquid medium 1 that is immiscible with a sample, for example, a microdroplet 3 of liquid paraffin injected from a micro nozzle 2, for example, a micropipette, at a focal position (+) of the laser beam. It is a microscope picture which shows the state which moves.
In this figure, a very small amount of liquid paraffin injected from the micropipette 2 into the water 1 with the injection pressure Pi and the injection time Ti forms an oil droplet 3 having a diameter of about 3 μm [FIG. 1 (a)], and further Pi. Is added for Ti seconds, and when 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)]. Then, the oil droplet 3 thus captured is moved to the lower left position [FIG. 1 (c)]. When Pi is added for Ti seconds and the oil droplet 3 ″ is emitted, the oil droplet is caused by the second laser beam. 3 ′ moves to the side of the oil droplet 3 [FIG. 1 (d)].
Therefore, if the micro container is arranged at the lower left position, the oil droplets 3, 3 ', 3 ",... Can be sequentially guided and accommodated therein.
[0009]
Next, FIG. 2 is an explanatory diagram in the case where different kinds of liquid samples are ejected from the two micro nozzles 2 and 2 ′ as micro droplets 3 and 3 ′ and collected in the micro containers 7 and 7 ′. Then, the liquid droplets 3 from the micro nozzles 2 and the liquid substrate 1 filled with the liquid medium 1 in which the liquid droplets 3 ′ from the micro nozzles 2 ′ are not mixed with the sample are processed to the upper portions of the micro containers 7 and 7 ′. It is emitted and captured by laser beams A and B, respectively. The droplets 3 and 3 ′ captured here are accurately moved by moving the focal positions of the laser beams A and B collected by the lens 8 three-dimensionally from the vicinity of the nozzle to the bottom of the micro container. It can be guided to the micro containers 7 and 7 'and collected.
[0010]
The ejection of liquid droplets from such a micro nozzle may occur due to the relationship between the properties of the liquid droplets to be ejected, the nozzle material, and the shape, and the liquid droplets after ejection adhere to the tip of the nozzle and cannot be reliably performed. In such a case, the ultrasonic wave is applied to the solution in the vicinity of the tip of the micro nozzle by the ultrasonic generating elements 5, 5 'such as a piezoelectric ceramic thin film formed or attached to the wall surface 4, 4' of the micro water tank. Dissociation and injection from the nozzle can be performed. The diameter and number of droplets to be ejected are changed by changing the ejection pressure, ejection time, and maintenance pressure.
[0011]
In the method of the present invention, instead of the liquid droplets, the fine particle-containing liquid droplets can be ejected from the micro nozzles 2 and 2 '. In this case, the solid sample and the liquid sample can be simultaneously guided and collected. Also, if the same kind of liquid droplets and medium droplets are lost at the same time the droplet portion away from fine nozzles, the same as the state in which only the fine particles that had been contained was released into the medium.
[0012]
Also, depending on the type of liquid sample to be collected, if the small container bottom by a hydrophilic or hydrophobic treatment, it is also possible immobilization after harvesting. Immobilization in the case of containing fine particles can be performed by a process such as ultraviolet ray irradiation after collecting ultraviolet curable droplets and fine particles in the same micro container.
[0013]
Note that to capture droplets of lower refractive index than the refractive index of the medium 1, and guided to the micro vessel, when accommodating uses a laser scanning manipulation method for scanning around the droplets.
[0014]
Next, FIG. 3 is a system diagram for explaining an example of a system configuration in which the focal positions of the two laser beams are guided three-dimensionally from the vicinity of the minute nozzle to the inside of the minute container, and accommodated . The laser light from the light source 10 is converted in diameter by the beam expander 11, passes through the half-wave plate 12, is decomposed by the first polarizing beam splitters 13 and 13 ', and passes through the electromagnetic shutters 14 and 14', respectively. The path is controlled by the focal position moving mechanisms 15 and 15 'configured by the galvanometer mirror pair and the focal position changing lens controlled by the computer Cp so that the focal position coordinates XYZ under the microscope become a desired position. Yes.
[0015]
The decomposed laser light is made coaxial again by the second polarizing beam splitters 16 and 16 ', and then introduced into the microscope 18 by the relay lens 17 so as to coincide with the axis of the epi-illumination light. The lens 8 collects and irradiates a spot size of about a wavelength size, for example, about 1 μm so as to be in the vicinity of the minute nozzle, thereby capturing the ejected droplet. Thereafter, as already described with reference to FIG. 2, the focal position moving mechanism 15, 15 ′ moves the focal position three-dimensionally to the position of the desired micro container 7 to accurately collect the droplet. Can do.
[0016]
At this time, the two laser beams do not interfere with each other because the polarization directions are orthogonal, and therefore the intensity distribution does not change depending on the relative position of the two beams, so that each laser beam is in a stable state. The target droplet can be captured and guided . Also, the movement path, movement speed, scanning position, scanning speed, synchronous scanning phase difference and scanning pattern of the focal position of the two laser beams indicate the state of droplet ejection, the capture by the laser beam, and the transport state by the CCD camera 19. , And the image is processed by the image processing apparatus 20, so that it can be automatically changed according to the purpose.
[0017]
In this way, in the method of the present invention, as shown in FIG. 1, not only the minute droplets sequentially ejected from one minute nozzle but also a plurality of minute nozzles as shown in FIG. Micro droplets ejected one by one or a plurality of micro droplets ejected one by one from a plurality of micro nozzles can be accurately guided and stored in a micro container.
[0018]
For these inductions , at least selected from the injection pressure, the injection time, and the maintenance pressure based on the data measured in advance while observing the state of the micro droplet ejected from the micro nozzle by image processing. By changing one factor, the diameter and number of microdroplets can be controlled.
[0019]
As described above, the method of the present invention has been described with an example in which a microdroplet or a microparticle-containing microdroplet is ejected into a liquid medium immiscible with the sample and the focal position of the laser beam is used. Similarly, when the microbubbles and the scanning center, the scanning trajectory, or the movement of the scanning cavity are used, they can be guided and accommodated in a predetermined micro container.
In this case, it is advantageous to collect microbubbles with the opening of the container facing downward and the bottom facing upward.
[0020]
【Example】
Next, the present invention will be described in more detail with reference to examples.
[0021]
Example 1
In the system shown in FIG. 2 or FIG. 3, a micro container formed by etching a microscope cover glass using a continuous wave Nd: YAG laser (wavelength 1064 nm, linearly polarized light) as a laser beam for capturing droplets is used as a bottom surface. A glass capillary is processed by irradiating two microscopic light beams from below through a microscopic water tank filled with distilled water (refractive index: 1.33) through a microscope oil immersion objective lens (magnification 100 times). The micro droplets (liquid paraffin, refractive index 1.46) ejected from the micro nozzle prepared in this way were captured and guided to the micro container.
That is, by controlling the injection pressure, injection time, and maintenance pressure with a pump from a micro nozzle, two droplets each having a size of 3 μm are ejected one by one, and each is captured by an optical tweezer method using two laser beams. FIG. 1 shows the result of observation with a microscope of the movement, guidance to the micro container, and accommodation .
[0022]
FIG. 1A shows a state after an injection pressure (3000 hPa) is applied to a minute nozzle for a set injection time (0.1 second), 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 focus position of the laser beam set near the nozzle tip in real time after image processing. FIG. 1B shows a state after the second injection pressure (3000 hPa) is applied for 0.1 second, a droplet at the nozzle tip is ejected and captured at the focal position of the laser beam at the white cross position. Shows the state. At this time, the next droplet is prepared at the nozzle tip. FIG. 1C shows a state in which the captured droplet is moved to the position of the micro container by computer control of the focal position moving mechanism 15 in FIG. In FIG. 1 (d), a second droplet is ejected by applying a third injection pressure (3000 hPa) for 0.1 second, and then the droplet is captured by the second beam. This shows a state in which the droplets are arranged in the X direction at positions adjacent to the droplets.
[0023]
Example 2
Using the same system as in Example 1, by changing the injection pressure, injection time, and maintenance pressure, two droplets with different diameters are ejected one by one, and each is captured by the optical tweezers method using laser light. , Moved and collected in a micro container. FIG. 4 shows a state where two ejected large and small droplets are captured by two beams and then arranged in the Y direction on the micro container. Thus, it can be seen that by controlling the injection pressure, the injection time, and the maintenance pressure, droplets having different sizes can be selectively ejected, moved precisely, and collected into the micro container .
[0024]
The present invention is not limited to the above-described embodiments, and the design can be changed as appropriate, such as the laser light introducing optical system, the positions, shapes, and number of the micro nozzles and the ultrasonic wave generating elements.
[0025]
【The invention's effect】
According to the present invention, it is possible not only to automatically extract a minute amount of liquid, gas and particulate reagent for constructing μ-TAS into a micron-sized array container, but also due to an excessive amount of reagent. Sensitive μ-TAS, micrometer-order chemical reaction fields, and microbial growth fields can be constructed without the occurrence of disturbance fields and contamination, and the reaction and growth can be controlled accordingly.
[Brief description of the drawings]
FIG. 1 is a photomicrograph showing a process in which a droplet ejected from a micro nozzle is collected into a micro container according to the present invention.
FIG. 2 is an explanatory diagram showing an example of a sampling method according to the present invention.
FIG. 3 is a system diagram showing an example of the configuration of the present invention.
FIG. 4 is a photomicrograph showing a state in which droplets having different diameters are ejected, moved , and collected in a micro container .
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Medium 2, 2 'Micro nozzle 3, 3' Micro droplet 4, 4 'Micro water tank wall surface 5, 5' Ultrasonic wave generation element 6 Transparent substrate material 7, 7 'Micro container 8 Condensing lens 9 Pump 10 Laser light source 11 Beam expander 12 Half-wave plate 13, 13 ′ First polarizing beam splitter 14, 14 ′ Electromagnetic shutter 15, 15 ′ Focus position moving mechanism 16, 16 ′ Second polarizing beam splitter 17 Relay lens 18 Microscope 19 CCD camera 20 Image processing apparatus

Claims (8)

In collecting a small amount of liquid or gas sample in a micro container, the sample is ejected from a nozzle into a liquid medium immiscible with the sample as micro droplets or micro bubbles, and the micro droplets or micro bubbles are focused on the laser beam. A method for collecting a small amount of a sample in a micro container, wherein the sample is captured at a position or a scan center, and is guided and accommodated in the micro container by moving the focal position or the scan center. When collecting a small amount of liquid or gas sample in a micro container, the sample is ejected as a micro droplet or micro bubble from a nozzle into a liquid medium immiscible with the sample, and the micro droplet or micro bubble is scanned with laser light. A method for collecting a small amount of a sample in a micro container, wherein the micro sample is captured in an orbit or in a scanning cavity, and is guided and accommodated in the micro container by moving the scanning orbit or scanning cavity.   3. The method for collecting a small amount of samples according to claim 1 or 2, wherein the micro droplets or micro bubbles are micro droplets or micro bubbles ejected one by one from a plurality of micro nozzles.   The method for collecting a small amount of sample according to claim 1 or 2, wherein the microdroplets or microbubbles are microdroplets or microbubbles sequentially ejected from one micro nozzle.   3. The method for collecting a small amount of a sample according to claim 1 or 2, wherein the micro droplets or micro bubbles are micro droplets or micro bubbles ejected in sequence from a plurality of micro nozzles per nozzle.   By observing the state of the micro droplet or micro bubble ejected from the micro nozzle by image processing, by changing at least one factor selected from the injection pressure, the ejection time, and the maintenance pressure, the micro droplet or micro 6. The method for collecting a trace amount according to claim 1, wherein the diameter and the number of bubbles are controlled.   The trace amount sampling method according to any one of claims 1 to 6, which is performed while applying an ultrasonic wave near the tip of a minute nozzle.   The trace amount sampling method according to any one of claims 1 to 7, wherein solid fine particles are contained in the fine droplets.

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