JP2014082975A - Droplet discharge apparatus for microorganism separation and/or arrangement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and an apparatus which recognize a position of droplet application in an absolute positional relationship, and make and land a minute droplet of microorganisms on a medium by a compact and simplified jetting mechanism without using a camera.SOLUTION: A droplet discharge apparatus according to the present invention discharges microorganisms onto a medium for separation and/or arrangement of the microorganisms, and comprises: a holding vessel for holding a liquid in which the microorganisms are dispersed; a discharge port which communicates with the holding vessel and discharges the liquid as droplets onto the medium; and an optical fiber having an opening for optical measurement in the proximity of the discharge port. A target point on the medium to which a discharged droplet is applied is determined by the light received by the opening of the optical fiber and then the droplet is jetted for application onto the medium.

Description

  The present invention relates to an apparatus for screening microorganisms using an ink jet or jet dispenser which is a system for jetting droplets, or an apparatus for sorting microorganisms, and means for individually separating and / or arranging microorganisms and Relates to the device.

  There are a huge variety and number of microorganisms that exist in nature. From these, useful microorganisms are searched for the development of pharmaceuticals, the production of food and chemical products, bioethanol, and bioremediation. It has been broken. The search (screening) process includes collection of microorganisms, pretreatment (impurity separation, concentration, dilution, dispersion in a culture solution), placement on a medium, culture, assay, and the like. These processes are mostly done manually and require a great deal of manpower, time and money.

In order to solve this problem, there is a method in which a microorganism can be separately applied in a micro liquid using an ink jet or a dispenser as a method capable of realizing a separation operation of a large number of microorganisms in a short time (Patent Document 1). . In this method, a minute liquid droplet of about 1 femtoliter to about 1 microliter is placed in a storage space having a size approximate to the liquid droplet, that is, the liquid droplet is applied to a concave portion of a flat plate on which unevenness is formed as a medium. It is necessary to arrange.
Ink jets that are widely used in printing devices, etc., can basically be applied to a flat surface such as paper or a flat plate so that the relative position is accurate in the overall arrangement of applied droplets. There is no prior art for a method that can start the droplet application position from a specific position and accurately determine the relative position of the entire droplet application arrangement. Therefore, even if the irregularities of the medium are regularly arranged and arranged, the position information of the end portions of the irregularities cannot be obtained, and the starting point for applying the droplet can be determined based on the positional information. Otherwise, the liquid droplets cannot be applied and arranged in the concave and convex portions. On the other hand, if the end portion of the concavo-convex portion can be positioned and the concave portion at the end can be set as the application start point, the relative position of the whole applied droplet can be set regularly by using an inkjet or the like, As a result, the droplets can be regularly arranged in the entire concave portion of the medium.

  In order to realize the accuracy of the application position of the inkjet or jet dispenser, there is a method for correcting the deviation from the target position when the droplet hits the target by observing the shape of the droplet and the flight trajectory of the droplet. It is disclosed (Patent Document 2). According to this method, a camera is provided for observing flying droplets, but the camera needs to have a function and a mechanism for capturing droplets flying at high speed. Specifically, the shutter is controlled in synchronization with the timing at which droplets are ejected, the camera is continuously shot to capture images of droplets flying in space at high speed, and in a short time. Since it is necessary to obtain image information, the exposure time of the camera is shortened, so the sensitivity of the camera is necessary, and an image photographing apparatus having such a function or mechanism is a jet dispenser that discharges droplets from the camera. There are wiring and control circuits that synchronize the discharge start control signal such as inkjet, etc., and it has a complicated mechanism, and since the exposure time of shooting of the camera is short, synchronous control to match the shutter control at high speed and timing is possible It is a complicated combination of a mechanical mechanism and an electronic control mechanism that can be performed precisely, and if a failure occurs even in a part of these complicated mechanisms, the function as a whole Impaired, as a result, the actual frequency of occurrence of malfunction or failure due to the operation is high. In addition, the image data, which is the image information of the droplets, has a large amount of data, requires a device and software for information processing for the purpose of a personal computer, etc., requires a device with high arithmetic processing capability, and also has software for analysis. Creation is required. A camera equipped with an optical mechanism has a larger device volume than an application device portion of an ink jet or a dispenser, and is arranged at a certain distance from the actual flight path of droplets. In addition, since the camera occupies a large space in the entire apparatus for applying droplets, there is a problem that the entire apparatus configuration becomes large. On the other hand, since the occupation of the camera is large, the application range may be limited.

  Although it is not a method for applying fine droplets, a method of using image analysis with a camera for application control of a dispenser is disclosed (Patent Document 3) for the purpose of applying in accordance with an uneven shape. Also in this method, application with a dispenser is observed with a camera, and the observation image is fed back and controlled. However, this method is not a method corresponding to the control of microdroplets, and using image analysis by a camera is the same method as the method using an ink jet.

JP 2005-137288 A JP 2005-205317 A JP 2009-172452 A

  In order to solve the above-mentioned problems of the prior art, the present invention recognizes the application position of a droplet in an absolute positional relationship, and uses a compact and simple mechanism to jet a droplet without using a camera. To provide a method and apparatus for landing microorganisms as microdroplets on a medium.

As a result of extensive studies, the present inventor has completed a droplet discharge device that separates and / or arranges microorganisms that have solved the above problems by using an optical fiber for optical measurement.
That is, the present invention is a droplet discharge device having the following characteristics.

[1] A droplet discharge device that discharges microorganisms onto a medium to separate and / or arrange microorganisms,
A holding container for holding a liquid in which microorganisms are dispersed; a discharge port that communicates with the holding container and discharges the liquid as a droplet onto a medium; and an opening of an optical fiber for optical measurement in the vicinity of the discharge port A droplet discharge device characterized by determining a target point on the medium for discharging droplets by light received from an opening of an optical fiber and discharging the droplets onto the medium by jetting the droplets .
[2] The droplet according to [1], wherein the optical fiber and the discharge port are provided so that an optical axis direction of the optical fiber and a discharge direction of the droplet are not parallel or orthogonal to each other. Discharge device.
[3] The optical fiber and the discharge port are provided so that the optical axis direction of the optical fiber and the discharge direction of the droplets are at an angle intersecting between the discharge port and the medium surface. 2].
[4] The droplet discharge according to any one of [1] to [3], wherein the optical measurement is performed by measuring any one of fluorescence, reflected light, transmitted light, and polarized light. apparatus.
[5] The droplet discharge device according to [4], wherein the optical measurement is performed with a single optical fiber.

According to the present invention, a device for performing microbial screening and microbial sorting does not require a high-performance camera, a mechanism for synchronization of photographing thereof, and an image analysis system, and a medium with a simple device configuration and an absolute positional relationship between micro droplets. Can land on top.
In particular, it is possible to reliably land the fine liquid droplets on the concave portion of the medium having an uneven shape.
In addition, it is possible to evaluate microorganisms whether microorganisms are growing or reacting with the same apparatus configuration.
Furthermore, it can be set as the apparatus which can confirm that the droplet was discharged from the droplet discharge port of the apparatus.

  In the case of performing droplet observation with a camera, in addition to the movement mechanism and fixing mechanism of the droplet discharge portion, the movement mechanism and / or fixing mechanism of the camera is necessary, whereas using an optical fiber. The moving mechanism and the fixing mechanism of the droplet discharge portion serve as the moving mechanism and the fixing mechanism of the optical fiber, so that the apparatus configuration is simplified, and a more space-saving and economically preferable apparatus configuration can be obtained. .

The figure which shows the droplet discharge apparatus which discharges the microorganisms using a jet dispenser on a medium, isolate | separates and / or arranges microorganisms. The perspective view of the medium which has an uneven | corrugated | grooved part. Sectional drawing of the medium which has an uneven | corrugated | grooved part. The schematic diagram explaining the transmitted light measurement of the convex part of a medium. The schematic diagram explaining detecting the transmitted light measurement of the recessed part part of a medium, and being a recessed part part. FIG. 4 is a schematic diagram for explaining that a medium is aligned with a droplet discharge port and a droplet is discharged into a concave portion of the medium. The schematic diagram explaining detecting the fluorescent label of the convex part of a medium. FIG. 4 is a schematic diagram for explaining that a medium is aligned with a droplet discharge port and a droplet is discharged into a concave portion of the medium. The schematic diagram explaining the reflected light measurement of the convex part of a medium. FIG. 6 is a schematic diagram for explaining that a concave portion of a medium is measured by reflected light to detect a concave portion (no reflection). FIG. 4 is a schematic diagram for explaining that a medium is aligned with a droplet discharge port and a droplet is discharged into a concave portion of the medium. The figure which shows the fluorescence measurement result of the fluorescence measurement of the medium hold | maintained in benzene atmosphere, and a control | contrast.

The droplet discharge device of the present invention includes a holding container that holds a liquid in which microorganisms are dispersed, a discharge port that communicates with the holding container and discharges the liquid as a droplet onto a medium, and is close to the discharge port. Equipped with an optical fiber opening for optical measurement, a target point on the medium for discharging droplets is determined by light received from the opening of the optical fiber, and discharged onto the medium by jetting droplets As a result, it is possible to accurately land the ejected droplets on the medium in an absolute positional relationship.

The method of jetting droplets used in the droplet discharge apparatus of the present invention is a droplet of a conventional inkjet method or jet dispenser method having a discharge port connected from a holding container holding a liquid in which microorganisms are dispersed. The method of jetting can be used. As a specific example of the ink jet system, an apparatus described in Patent Document 1 can be given. Examples of the driving method of the ink jet include a driving method using a piezoelectric element such as piezo and a jet method using bubbles generated by rapid heating of the heating unit, but does not heat the droplets. The piezo method is preferred.
Examples of the jet dispenser include a method of releasing the electromagnetic valve for a short time while increasing and maintaining the pressure in the holding container that holds the liquid by the gas having the pressure from the compressor or the cylinder. The jet dispenser is preferable because it can change the volume of the ejected liquid droplets depending on the pressure in the holding container, the release time of the electromagnetic valve, and the opening diameter of the ejection port, and does not heat the liquid droplets.

  By using the above-described droplet discharge device, a large number of minute droplets can be discharged from a discharge port at high speed onto a medium. The volume of the discharged droplet is usually preferably 1 femtoliter (fl) to 1 microliter (μl), more preferably 1 picoliter (pl) to 100 nanoliters, most preferably 10 picoliter to 10 nanoliters. It is better to discharge in liters. The volume (particle diameter) of the droplet can be controlled by controlling the inner diameter of the nozzle of the discharge port and the pressure applied to the liquid by the pressure generating member.

A medium that is a target of droplets ejected from the droplet ejection apparatus of the present invention has a flat shape. The shape of the flat plate is preferably a rectangle, but may be a disk, an ellipse, or a polygon such as a triangle.
As the material, glass, ceramics, metal, silicon, quartz, plastic, or the like can be used. Among them, a material that can handle sterilization is preferable, glass or plastic that can be steam sterilized is preferable, or plastic that can transmit gamma rays is preferable for gamma sterilization, or dry. In heat sterilization, glass, metal, quartz, and ceramics that can be heat-treated at about 150 ° C. are preferable.

The surface shape of the medium may be a concavo-convex shape, or may be a flat state having no concavo-convex shape as long as each droplet can hold the droplet independently after landing. In the present invention, since a large number of droplets can be ejected from the droplet ejection device onto the medium at a high speed, a large number of concave and convex shapes are provided regularly or irregularly on the concave-convex medium. ing. The number of the concave portions on the medium can be arbitrarily set depending on the size of the medium, the interval between the concave shapes, and the size of the concave shapes, but is usually several thousand or more.
An example of the case where the concave / convex shape of the medium is provided in a regular geometric shape can use lithography technology, etc., and the interval between the concave portion in the medium and the adjacent concave portion is the same as the other concave portions in the medium. It can be made the same as the interval between the adjacent concave portions.
When ejecting droplets onto a flat medium having no uneven shape, a medium having a hydrophobic (water-repellent) region and a hydrophilic region described in the cited document 1, a charged region and a non-charged region are used as the medium. Any of a medium having an electrode and a medium having an electrode formed in a predetermined pattern can be used.

  The ejection of droplets from the droplet ejection device onto the medium may be performed by independently moving at least one of the ejection head and the medium in any direction. It is performed by moving at least one of them independently in two orthogonal directions.

  Typically, it is a so-called XY plotter. As an example of the embodiment, a configuration in which the ejection head is moved in the X direction and the medium is moved in the Y direction can be exemplified, but the ejection head may be moved in the Y direction and the medium may be moved in the X direction. Of course, any one of the ejection head 4 and the medium 1 may be moved in two directions of X and Y. Since the configuration of the XY plotter is well known, detailed description thereof is omitted.

  The droplet discharge device of the present invention is equipped with an optical fiber opening for optical measurement in the vicinity of the discharge port. The optical fiber is equipped so that the optical axis direction of the optical fiber and the discharge direction of the droplets are at an angle that is not parallel or orthogonal.

Furthermore, the optical fiber and the ejection port may be provided so that the optical axis direction of the optical fiber and the ejection direction of the droplets are at an angle that intersects between the ejection port and the medium surface.
In this case, it is possible to have a feature that it can be confirmed by optical measurement of the optical fiber that the droplet is discharged from the droplet discharge port of the apparatus.

  The light measured by the optical measurement of the droplet discharge device of the present invention includes fluorescence, reflected light, transmitted light, or polarized light.

In the case of optical measurement of fluorescence, the medium is irradiated with excitation light, and the fluorescence generated by the excitation light is picked up by an optical fiber and measured. When optically measuring reflected light, the medium is irradiated with light from a light source that generates the reflected light, and the reflected light reflected from the irradiated light is picked up by an optical fiber and measured.
When optically measuring transmitted light, a light source is placed on the opposite side of the medium as viewed from the optical fiber side, and the transmitted light, which is light transmitted through the medium, is picked up by the optical fiber and measured.

  When optically measuring the polarization, a light source and a polarizing filter are placed on the opposite side of the medium as viewed from the optical fiber side, and the medium is irradiated with light that has been polarized through the polarizing filter. By laying a polarizing filter at the light entrance of the optical fiber so as to be orthogonal to the polarized light, the polarized light that irradiates the medium cannot be detected by the optical fiber, but there is a birefringent property substance on the surface or concave portion of the medium. For example, the plane of polarization of the light from the light source side changes and passes through the polarization filter on the optical fiber side. As a result, the transmitted light can be picked up and measured by the optical fiber.

  In the present invention, an optical fiber opening is provided in the vicinity of the discharge port of the discharge mechanism of an ink jet or jet dispenser, and a mark on the medium or a concave portion or a convex portion of the medium is optically measured with the optical fiber. It is characterized in that a discharged droplet is landed on a medium.

  An optical fiber is fixed in the vicinity of the ejection port of the ejection mechanism of the inkjet or jet dispenser, and the relative position between the optical axis of the optical fiber and the droplet ejection port of the inkjet or dispenser is determined in advance. If the position of the concave portion can be measured, the information on the relative position can be used to easily move the droplet discharge port to a position where the droplet discharge port can land the droplet on the concave portion accurately. Then, by moving the droplet discharge port to an appropriate position, the droplet can be discharged and the minute droplet can be accurately landed on the concave portion.

In the case of a medium in which the medium is a concavo-convex flat plate, the ejected liquid droplets can be accurately landed on the concave position by optically measuring the mark on the medium or the concave or convex part of the medium with an optical fiber.
In other words, a sign is laid in advance so as to cause a change in the optical signal by optically measuring the concave position of the medium directly, or on a part of the convex shape of the medium spaced from the concave position of the medium. In addition, by optically measuring the marked portion with an optical fiber, the ejected liquid droplet can be accurately landed on the concave position.
In the case of the method of recognizing the concave position by the sign, if there is information on the relative position of the sign and the uneven portion, the position information of the uneven portion can be determined by detecting and recognizing the sign.

  The label causes the light to be physically changed such as light intensity, illuminance, refraction, and polarization so that optical measurement using an optical fiber can be performed, or the light energy is converted and the wavelength is changed like a fluorescent substance. It is a landmark that can.

When the optical measurement method is fluorescence, any material medium can be used, but it is desirable to use glass or metal with low autofluorescence as the medium, and a fluorescent substance that emits fluorescence on the medium is used as a label. Lay it down. Here, as the means for laying the fluorescent substance, the fluorescent substance is applied to the surface of the medium, the fluorescent substance sheet is attached, the fluorescent substance dissolved liquid is sprayed, or the fluorescent substance is applied to the ink or the adhesive. Kneading and printing or coating. The fluorescent substance as a label on the surface of the medium may be laid as a point, or may be laid in a linear shape, or may be laid as a geometric pattern, or on the entire surface of the convex and concave portions. May be laid.
In addition, if the medium is made of a transparent material and does not absorb excitation light, or is a material that transmits light of a fluorescence wavelength, the fluorescent material can be seen on the medium surface on the back side when viewed from the end face of the optical fiber. May be laid.
Furthermore, if the medium is a material that can transmit light and can transmit the excitation wavelength and fluorescence wavelength of fluorescence, fluorescence can be measured using not only reflected light but also transmitted light.

When the optical measurement method is reflected light, optical measurement is performed by causing a signal difference of the reflected light by causing the surface of the medium to have a mirror-like or diffusely reflected state.
If the surface of the medium is a material that easily reflects light, such as metal, or a medium whose surface is mirror-finished, the surface of a part of the medium can be roughened to scatter incident light, and the label can be reversed. In addition, if the medium is in a surface state in which light incident on the surface of the medium is scattered, such as ceramic, the mark portion is subjected to vapor deposition treatment by mirror treatment, metal plating treatment, sputtering, etc. It can be processed and labeled to make it efficient. Any medium made of any of the materials described above can be used as long as the surface can be made into a mirror surface or an irregular reflection surface.

If transmitted light is used for optical measurement, if the medium is made of a material that can transmit light, a method for detecting the transmitted light, that is, the target portion can be detected using the intensity of light transmitted through the medium as an index. it can.
Further, if the medium is a material that transmits light and is made of a material that maintains a specific refractive index, the target portion can be detected by utilizing the refraction of light incident on the medium.
When visible light is used as transmitted light, it is desirable to use plastic, glass, quartz, or the like that is permeable to the medium. In addition, when ultraviolet light is used as transmitted light, it is preferable to use quartz having transparency as a medium.

By detecting the difference in transmitted light intensity between the concave and convex portions of the concave and convex portions, the position information of the end portions of the concave portions can be determined.
Further, if the medium is a material that transmits light and is made of a material that maintains a specific refractive index, the uneven portion can be detected by using the refraction of light incident on the medium. Light incident from the opposite side of the uneven portion of the medium travels from the surface of the medium to the inside of the medium with a refractive index, but due to the difference in the thickness of the medium material of the uneven portion, Since the path of the light exiting to the outside changes, the uneven portion can be detected by detecting the difference.

  It is also possible to use a medium having a flat surface without an uneven portion. For example, by printing on a flat surface of the medium and creating a portion that does not transmit light on the surface of the medium, the portion where the droplets land can be delimited. If metal deposition is performed by using a photomask and a resist instead of printing, detection can be performed using transmitted light, or reflected light can be used.

  If the polarized light is used for optical measurement, the light transmitted through the medium can be polarized light, and a substance having polarization characteristics such as a polarizing film or a polarizing plate on the surface of the medium can be used as a label for the medium. More specifically, if the plane of polarization of the light transmitted through the medium and the plane of polarization of the substance laid on the label portion of the medium surface are set perpendicular to each other, only the label portion does not transmit or transmit light. The label portion can be detected by reducing the intensity of the light.

  The light source for optical measurement may be an LED, laser diode, halogen lamp, mercury lamp, or light bulb, which can irradiate the medium, and the optical fiber detects the difference in optical signal between the marker or the concave portion and the convex portion. I can do it.

  When the medium is irradiated with light from the discharge nozzle side, the light from the light source is preferably irradiated through an optical fiber, and it is preferable that the irradiation light and the detected light are transmitted through a single optical fiber.

Moreover, it is preferable that the end surface of an optical fiber is equipped with the condensing optical system.
The light condensing optical system on the end face can collect the light passing through the optical fiber outside the end face, the focal distance that is the distance between the end face and the focal point is determined, and the distance between the end face of the optical fiber and the target medium The distance between the end face of the optical fiber and the medium can be measured using the change in the optical signal caused by changing.
If the information on the relative spatial position of the end face of the optical fiber and the droplet discharge port is obtained in advance, the distance between the droplet discharge port and the medium can be grasped based on the measurement information of the distance between the optical fiber and the medium. .

  Droplets that pass through the optical axis of the optical fiber by equipping the optical fiber and the discharge port so that the optical axis direction of the optical fiber and the discharge direction of the droplet intersect at an angle between the discharge port and the medium surface However, by receiving the light being measured by the optical fiber, the light beam being measured becomes discontinuous, and by detecting the optical signal by the optical fiber, it can be detected that the droplet is being ejected.

In the present invention, the optical measurement function of the optical fiber of the present invention can be used independently of the droplet discharge function, and it is possible to determine whether microorganisms to be screened are growing or reacting in each concave portion of the medium. The evaluation can also be performed using the difference in the optical signals of fluorescence, reflected light, and transmitted light.
In order to sort the microorganism cells, the microorganisms can be individually arranged on a medium having irregularities, and the characteristics of the microorganisms can be evaluated by optical measurement.

  Hereinafter, a liquid droplet ejection apparatus according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings using an example of a jet dispenser.

  FIG. 1 is a schematic perspective view of a droplet discharge device 100 that discharges microorganisms using a jet dispenser onto a medium to separate and / or arrange microorganisms. As shown in FIG. 1, a dispenser comprising a dispenser head unit 1, a tube 24 for transmitting air pressure to the head unit, a solenoid valve control code 25 for the head unit 1 and a solenoid valve opening / closing controller 23 ejects micro droplets. The optical fiber 3 is connected to a condensing device 4 at the end of the optical fiber, which is an optical system that condenses the light at the center of the optical axis, and the condensing device 4 is connected to the dispenser head 1 of the dispenser. It is laid and placed.

The dispenser head portion 1 is filled with the liquid to be discharged, and the electromagnetic valve opening / closing controller 23 opens and closes the electromagnetic valve incorporated in the dispenser head portion 1, thereby causing the liquid droplets with the air pressure of the tube 24. Are discharged.
The distance between the discharge port of the discharge nozzle 2 of the dispenser 1 and the optical axis of the optical fiber is preferably within 10 cm, more preferably within 2 cm, and even more preferably within 5 mm. Any distance may be used as long as the discharge outlet can discharge the droplet and the optical fiber can detect the medium mark as described above.

  The dispenser head unit 1 and the light collecting device 4 at the optical fiber end are connected to an XYZ mobile robot 5. By moving the dispenser head unit 1 and the condensing device 4 at the end of the optical fiber by the robot 5 in the X-axis direction and the Z-axis direction, and moving the medium 6 by the robot 5 in the Y-axis direction, the movement of the XYZ axes can be performed. Do. In the drawing, the dispenser head unit 1 and the light collecting device 4 at the end of the optical fiber are illustrated as being moved by the robot 5 as an integrated device, but may be configured to move separately.

  The dispenser 1 can communicate with the PC and communicate information 22 as communication with a personal computer (PC: not shown), and the droplet discharge is controlled by the dispenser. The XYZ mobile robot 5 can also control and communicate information 22 with the PC, and the movement control of the X-axis and Z-axis is performed on the dispenser head unit 1 and the condensing device 4 at the optical fiber end.

  A light source device for sending excitation light for irradiating a medium to an optical fiber and a detector 21 for optical fiber guided light from the medium side send out light of a specific wavelength to the optical fiber 3 and pass through the condensing device 4 at the end of the optical fiber. Is irradiated with light. The fluorescence generated by the irradiation is sent to the optical fiber 3 through the light collecting device 4 at the end of the optical fiber, and the light source 21 that sends the excitation light for irradiating the medium to the optical fiber 3 and the optical fiber waveguide light detector from the medium side. Guided to 21. A light source for sending excitation light for irradiating the medium to the optical fiber and a detector 21 for the optical fiber guided light from the medium side measure the intensity of the fluorescence guided through the optical fiber 3.

  The fluorescence intensity signal is transmitted to the PC as digital information through control and information communication 22 with the PC. Further, the control of the light source device that sends the excitation light for irradiating the medium to the optical fiber and the optical fiber waveguide light detector 21 from the medium side is performed through the control 22 and information communication 22 with the PC.

FIG. 2 shows an example of a medium having an uneven shape on a rectangular flat plate as an example of the medium. As shown in FIG. 2, the medium 6 is a flat plate formed such that the concave portions 7 are regularly arranged.
FIG. 3 is a diagram showing a cross-sectional portion of the medium, and shows an uneven-shaped cross section 9 of the medium 6 along a dotted line 8 in FIG.

4, 5 and 6 describe a method of measuring transmitted light.
As shown in FIG. 4, the light source light 11 from the light source 10 irradiates the convex portion of the medium 9 or a portion having the same thickness as the convex portion, and the light 12 transmitted through the convex portion as the transmitted light is applied to the optical fiber 3. Light is collected and detected by the condensing device 4 at the end of the connected optical fiber. As the light source 10, an LED is illustrated in FIG. 4, but the light source 10 is not limited, and a light source such as a semiconductor laser, a halogen lamp, a mercury lamp, or a fluorescent lamp can be used.

As shown in FIG. 5, when the medium is moved in parallel so that the light irradiation portion from the light source 10 is moved so that the concave portion is irradiated, the light intensity transmitted through the concave portion is relatively It can be detected that the illuminance is improved as an optical signal, and that the optical axis of the optical fiber 3 is within the range of the concave portion of the medium.
If the position of the concave portion can be detected as shown in FIG. 5, then the medium 9 is moved as shown in FIG. 6 by the length of the distance from the optical axis position of the optical fiber 3 to the discharge port position of the discharge head 2 of the dispenser. By doing so, the position of the concave portion of the medium can be aligned with the discharge port of the liquid droplet, and the droplet 14 can be discharged so as to land on the concave portion.

  The medium can be formed into a regular geometric shape by using a lithography technique or the like, and the distance between the concave portion in the medium and the adjacent concave portion is equal to the other concave portion in the medium and the adjacent portion. It can be made the same as the interval between the concave portions. In such a medium, if the concave portion at the extreme end of the concave / convex portion of the medium can be detected, the medium is moved so that the droplet discharge port coincides with the concave portion if the medium is moved at a certain moving distance. It is possible to repeat the operation for the number of recesses by discharging one drop of liquid with one movement, then performing one movement and discharging one drop of liquid. In this case, it is possible to correctly eject and land the droplet on the concave portion of the medium.

  Even if the medium is not geometrically regular irregularities, by measuring the transmitted light intensity of the convex part and the transmitted light intensity of the concave part while moving the medium by a distance of a certain length, By detecting the position of the concave portion and moving the medium by the length of the distance between the optical axis of the optical fiber and the droplet discharge port of the dispenser, the concave portion can be accurately identified. Subsequently, in order to discharge the droplets, the medium is moved in the original position direction by the distance moved immediately before, and the operation of measuring the transmitted light is repeated in order. The liquid droplets can be landed on the concave portions and the liquid droplets can be landed on the concave portions of the entire medium.

Even if the medium is a material that does not transmit light, as shown in FIG. 7, an optically identifiable marker is laid on the convex portion of the medium or a portion having the same thickness as the convex portion. The position can be detected.
The label may be anything as long as it can emit an optical signal that can be optically measured using an optical fiber. In FIG. 7, a label 17 having fluorescence characteristics is applied to a medium using a fluorescent substance as a label. An example is shown. In FIG. 7, excitation light 15 for exciting the labeled fluorescent substance is irradiated from the optical fiber 4, and fluorescence 16 generated by the excitation light is detected by the optical fiber.
If the distance between the marked portion of the medium and the concave portion at the end can be set or measured in advance, first, the marked portion of the medium and the extreme end are adjusted so that the optical axis of the optical fiber is within the range of the concave portion. By moving the medium by the distance from the concave portion of the liquid, and then moving the medium by the distance between the optical axis of the optical fiber and the droplet discharge port of the dispenser, the droplet discharge port is The droplet 14 that can land on the concave portion at the end is ready to be discharged. If the medium is a concave / convex shape with a geometrically constant pattern in the adjacent concave portion, a droplet discharge port is arranged in the adjacent concave portion if the medium is moved by the distance between the adjacent concave portions. be able to.

  The fluorescent substance as a label on the surface of the medium may be laid as a point, or may be laid in a linear shape, or may be laid as a geometric pattern, or on the entire surface of the convex and concave portions. May be laid.

If the concave portions are regularly arranged on the medium, for example, in a matrix state, the position information can be detected by detecting the transmitted light or the like at the concave portions at the ends.
Alternatively, in addition to the concave portions regularly arranged as line examples, the concave portions may be formed on the medium as marks.

  Even if the medium is not geometrically regular uneven shape, if a marker capable of optical measurement can be placed on the surface of the convex part around the concave part, it is adjacent by performing optical measurement while moving the optical axis of the optical fiber A liquid droplet can be discharged and landed on the concave portion of the medium while crossing the concave portion.

9, 10, and 11 show a case where reflected light is measured. If a sign 18 having a property of reflecting light is laid on a medium to be used as a sign, the light transmitted from the optical fiber 3 to that portion is irradiated onto the medium via the light collecting device 4 at the end of the optical fiber. 19 is irradiated on the sign 18 having the reflection characteristic, and is picked up and measured as reflected light 20 through the light collecting device 4 at the end of the optical fiber. Subsequently, when the medium is moved while measuring the reflected light, the concave portion on the most end side of the medium moves so that the optical axis of the optical fiber coincides, and as shown in FIG. The light 19 irradiating the medium through the optical device 4 passes through the medium, and as a result, the reflected light is not measured. Subsequently, by moving the medium by the distance between the optical axis of the optical fiber and the droplet discharge port of the dispenser, the droplet discharge port causes the droplet 14 that can land on the concave portion at the end as shown in FIG. It will be in the state which can discharge.
If the medium is a concave / convex shape with a geometrically constant pattern in the adjacent concave portion, a droplet discharge port is arranged in the adjacent concave portion if the medium is moved by the distance between the adjacent concave portions. be able to.

  Examples of the present invention are illustrated below, but the present invention is not limited to these examples.

Example 1
A jet dispenser CyberJet 2 made by Musashi Engineering was used as the dispenser, and an XYZ-driven desktop robot SHOTMASTER-DS made by Musashi Engineering was used as the XYZ moving robot for moving the dispenser head.
Fixing the light collecting end face of the optical fiber via the jig fixing the dispenser head to the robot, and integrating the dispenser head with the optical fiber, the optical fiber is connected to the discharge port of the jet dispenser. The openings of were close.

  The optical fiber was connected to a fluorescence detector FLE1100 manufactured by Nippon Sheet Glass Co., Ltd., and the excitation light was guided and the fluorescence was guided by a single optical fiber. The excitation light wavelength of the used apparatus is 470 nm, and the detection fluorescence wavelength is 530 nm. An optical fiber having an optical fiber end condensing device connected to the tip of the optical fiber was used.

  Since the light source of the excitation light is an LED, its wavelength spectrum has a long wavelength side and a short wavelength side centering on the excitation light wavelength of 470 nm, and the wavelength spectrum extends to the vicinity of the detection fluorescence wavelength of the fluorescence detection device. The skirt part is faint but spreads. In actual device measurement, a minute signal can be captured when the focal point of the condensing device at the end of the optical fiber is brought close to a surface where light reflection occurs, and a minute signal change can be captured based on the presence or absence of a reflecting object. By detecting this minute signal change, the fluorescence detection apparatus can detect a portion having a difference in the reflection characteristics of the medium.

  A total of 6400 holes, 80 horizontal and 80 vertical, were formed in a 5 cm square transparent glass material to obtain a medium. The size of one hole of the medium was a diameter of 400 μm, a center depth of 200 μm, and the distance between the holes was 200 μm.

  For labeling the medium, a riboflavin that is excited by blue excitation in a silicon adhesive and kneaded is prepared, and a circle having a diameter of 400 μm is formed at a position 200 μm away from the center of the hole corresponding to the concave portion at the end of the medium. It was deposited as a shape and cured.

  The acrylic plate was processed to prepare a guide for fixing a 5 cm square glass medium, and fixed using a screw hole in a movable stage of an XYZ mobile robot.

  The control of the XYZ mobile robot and the optical signal measurement operation of the light source device that sends the excitation light for irradiating the medium to the optical fiber and the optical fiber waveguide light detector from the medium side were controlled by a program of a personal computer.

  The serial communication RS232C cable is used to perform serial communication for transmission of commands for driving control of the XYZ mobile robot and serial communication for reception of position information of the portion connecting the robot and the dispenser head. A connection was made between the communication terminal and the robot communication terminal. In addition, in order to transmit optical signal information to a personal computer, a serial communication RS232C cable was used to connect between the communication terminal of the personal computer and the communication terminal of the fluorescence detector.

  The moving speed of the robot arm was 200 mm per second.

  In the personal computer program, the part that connects the robot and the dispenser head is moved to the vicinity of the position where the mark on the medium is located. The data which became the pair of optical signal information and position information was acquired. Next, based on the position information where the change of the optical signal was observed, the position information where the change of the optical signal was observed was moved in the X-axis direction. Next, the optical signal was measured while moving in the Y-axis direction at a pitch of 10 μm, and position information where the change of the optical signal occurred was obtained. With the above operation, the XY position information of the label position of the medium is obtained.

  Based on the label and the position information, the droplet is moved to the concave portion located at the end of the medium, and the jet dispenser is driven to discharge a droplet so that one droplet contains one cell microorganism. Then, a droplet was landed on the concave portion at the extreme end in the medium. Subsequently, the droplet is moved in the X-axis direction so that the droplet can be discharged at the position of the adjacent concave portion on the medium. Similarly, the droplet is discharged, and this operation is repeated. This was repeated until the number of concave portions in the medium was discharged. Subsequently, while moving in the Y-axis direction and moving in the opposite direction of the X-axis, the liquid droplets are ejected to the concave portions, and the operation is performed to eject the number of concave portions in the medium in one line in the X-axis direction. Repeat until This operation was repeated until the liquid droplets could be discharged into the concave portions of all the rows on the medium. Through this series of operations, the droplets could be arranged in the concave portions on the medium.

(Example 2)
For the actual microorganism separation operation, the method described in Example 1 was used. The target microbial solution to be discharged as droplets was obtained by suspending soil in a solution in which fructose and tetrachloroauric (III) acid were mixed in a buffer solution containing an inorganic salt, followed by 10 μm filter filtration.

  The buffer containing the inorganic salt is a solution containing 5% solution A, 20% solution B, 0.1% SS solution, 0.05% polypeptone and 0.025% dry yeast extract in sterile distilled water. The composition of solution A is a solution in which 20 g of ammonium sulfate, 2.465 g of magnesium sulfate, 0.0278 g of iron sulfide heptahydrate, 0.147 g of calcium chloride dihydrate, and 5 g of sodium chloride are dissolved in a 500 ml solution. is there. Solution B is a 500 milliliter aqueous solution containing 35.8 g of disodium phosphate monohydrogen dodecahydrate and 13.61 g of potassium monophosphate dihydrogen. The composition of the SS solution is as follows: in 500 ml volume, zinc sulfate heptahydrate 0.201 g, ammonium molybdate tetrahydrate 0.015 g, copper sulfate pentahydrate 0.02 g, cobalt chloride hexahydrate 0 An aqueous solution containing 0.04 g and manganese sulfate pentahydrate 0.149 g.

  The above sample was observed under a microscope, and the number of microorganisms was counted to estimate the concentration of microorganisms in the liquid. After preparing so that one microbial cell may contain in the volume of one droplet of 400 micrometers in diameter discharged with a jet dispenser, the droplet was discharged with respect to the medium with the jet dispenser. The ejection of the liquid droplets onto the medium having 6400 concave portions was completed in 30 minutes.

  After discharging droplets on the medium, the sample was kept at room temperature for 2 days, and then optically measured as reflected light with the system of the present invention. In the optical measurement result of the concave portion of the medium, the concave portion where the signal is different from other concave portions was detected.

  It is considered that there are microorganisms that can reduce tetrachlorogold (III) acid and convert it to metal gold in the concave portion, and the liquid taken out from the concave portion is mixed with inorganic salt, fructose, tetrachlorogold (III) acid. After inoculating the sterilized medium containing and maintaining at room temperature for 5 days, a purple color indicating that tetrachloroauric (III) acid was reduced and precipitated as gold nanoparticles as the microorganisms grew. A color was observed.

  According to the present invention, microorganisms that reduce gold ions can be separated in a short time.

(Example 3)
The oil-contaminated soil was suspended in the same inorganic salt buffer as that used in Example 2 to prepare a soil suspension, and filtered through a 10 μm filter. A diluted sample of a 10-fold dilution series of the filtrate was prepared using an inorganic buffer, stained with the fluorescent staining reagent acridine orange, and the number of microorganisms in the solution was counted by observing with an Olympus BX60 fluorescence microscope.

  The filtrate is diluted with an inorganic salt buffer solution to the concentration of one microbial cell contained in the volume of one droplet having a diameter of 400 μm discharged by the jet dispenser, and the droplet is applied to the medium with the jet dispenser. Was discharged.

  After 6400 droplets are applied to a glass medium, the glass medium in which the droplets are arranged is placed in an anero-pack square jar, which is a plastic container that can be sealed, and a benzene solution is placed in a small glass container. The lid was sealed and sealed, and kept at room temperature for 5 days. It is considered that benzene has a low vapor pressure and is vaporized in a sealed container, so that benzene is dissolved in droplets corresponding to the concave portions of the medium. As a control, a glass medium similarly coated with droplets was sealed without benzene in an aneropack square jar.

  After sealing and opening the lid after 5 days, the glass medium was taken out, and fluorescence detection with an optical fiber was performed on 6,400 droplets on the medium. Microorganisms biosynthesize and encapsulate riboflavin as a vitamin component in cells, and generate blue-green fluorescence by blue light. If there is a microorganism that can decompose and use benzene as an organic substance among microorganisms in the soil, it grows using benzene and increases the number of cells. If the number of microbial cells increases, a blue-green fluorescence signal is strongly generated by blue light irradiation.

  As an example of fluorescence measurement, the result of fluorescence measurement of 30 droplets is shown in FIG. Compared with the control, it was measured that the fluorescence intensity of the concave portion in the medium sealed under the benzene atmosphere was higher, and it was shown that the microorganisms were growing in the concave portion in the medium. That is, it was shown that microorganisms that can utilize benzene as an organic substance are inherent in the contaminated soil.

  By using the present invention, it is expensive, occupies the device space, and eliminates the need for high-performance camera photography that restricts the operating range of the dispenser and the inkjet, and it is possible to perform screening of microorganisms and sorting of microorganisms with a simple mechanism. This makes it possible to reduce the costs of the microorganism screening device and the microorganism sorting device, or to achieve compactness and space saving.

  Due to such effects, the search for microorganisms can be promoted on a general basis, and the effective use of microbial functions is expanded, so innovative use of microorganisms is expected in manufacturing processes, etc., or it has been unknown until now. Bioactive ingredients can be easily searched for in a short period of time, and it is expected to contribute to the development of pharmaceuticals effective for the treatment of various diseases and the development of biofuel and bioplastic manufacturing technologies.

  In addition, it can be applied to microbial sorting with a simple machine configuration, and there is a possibility that microbial sorting can be handled near the treatment site in the management process of fermentation processes, bioremediation and wastewater treatment involving complicated microorganisms, Proper processing is considered to bring about economic benefits by shortening the processing period.

1: Dispenser head unit 2: Discharge nozzle 3: Optical fiber 4: Condensing device 5 at the end of the optical fiber: XYZ mobile robot 6: Medium 7: Recessed portion of the medium 8: Dotted line 9 indicating the cross-sectional portion of the medium 9: Section 10: Light source 11: Light 12 of the light source 12: Light transmitted through the convex portion 13: Light transmitted through the concave portion 14: Discharged droplet 15: Excitation light 16: Fluorescence 17: Marked object 18 having fluorescence characteristics: Reflection characteristics Marked object 19: Irradiation light 20: Reflected light 21: Light source device for sending excitation light for irradiating the medium to the optical fiber and optical fiber waveguide light detector 22 from the medium side: Control with PC and communication of information 23: Electromagnetic Valve opening / closing controller 24: Tube for transmitting air pressure to the head 25: Electromagnetic valve control code 100: Droplet discharge device

Claims (5)

A droplet discharge device that discharges microorganisms onto a medium to separate and / or arrange microorganisms,
A holding container for holding a liquid in which microorganisms are dispersed; a discharge port that communicates with the holding container and discharges the liquid as a droplet onto a medium; and an opening of an optical fiber for optical measurement in the vicinity of the discharge port A droplet discharge device characterized by determining a target point on the medium for discharging droplets by light received from an opening of an optical fiber and discharging the droplets onto the medium by jetting the droplets 2. The droplet discharge device according to claim 1, wherein the optical fiber and the discharge port are provided so that the optical axis direction of the optical fiber and the discharge direction of the droplets are at an angle that is not parallel or orthogonal. The optical fiber and the discharge port are provided so that the optical axis direction of the optical fiber and the discharge direction of the droplets are at an angle that intersects between the discharge port and the medium surface. Droplet discharge device The liquid droplet ejection apparatus according to claim 1, wherein the optical measurement is performed by measuring any one of fluorescence, reflected light, transmitted light, and polarized light. The liquid droplet ejection apparatus according to claim 4, wherein the optical measurement is performed with a single optical fiber.