JP2011120582A - Automated apparatus for component capture, injection, and molecular analysis accompanied by observation of microregion such as cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve such a problem that guiding of a nano-spray ionization capillary tip to a target position such as a cell in the microscopic visual field with only the visual field of a minute region is a process most requiring labor, time and techniques, and becomes a bottleneck in a method for measurement when a sample of the minute region such as one living cell is sucked and captured with the nano-spray ionization capillary tip while carrying out microscopic observation to perform direct mass spectroscopy. <P>SOLUTION: The sample capturing capillary such as the nano-spray ionization capillary tip for piercing the cell etc., under a microscope is held at the end of a robot, and the tip position of the tip thereof is detected and corrected to correct the space coordinates of the tip position of the tip for each tip mounting. Furthermore, a focus plane position for observing the coordinates for the space of a focal position under the microscope is corrected with Z-axis encoding of a microscopic stage to guide the tip between both the points with the robot. Thereby, the sample capturing capillary is guided to the object minute sample such as the cell at a high speed with high accuracy. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a solution capturing method and a component analysis method in a micro space such as a cell accompanied by observation of a micro space body such as cell observation, and a micro space liquid capturing and analyzing apparatus such as a cell fluid.

The molecular mechanism in a cell is an essential one that causes a life phenomenon, and is an eternal issue that should be elucidated by life science. However, until now, there has been no analysis method for searching for molecules in living cells in real time and elucidating the mechanism. If such a method or device is developed, it will be possible to quickly find a candidate molecule that can be a marker molecule or a drug for detecting not only a life phenomenon but also a disease state or a disease state, and the benefits of mankind are immeasurable.
In general, in such an analysis, conventionally, a cell is placed in a set state, and a large number of cells before and after setting conditions such as external factors such as stimuli are collected and ground. Such molecular analysis is performed separately, and the molecular mechanism of living organisms is estimated from the results of molecular search that can be said to be the average value of many cells. However, we recognize from years of video microscopy of cells that cell responses are not the same.
In recent years, with the advancement of nanotechnology, while observing changes in substances in the submicrometer range of 200 μm or less, the molecular change is finally detected by, for example, mass spectrometry or other microanalysis methods. It is also necessary to grasp it together with the actual state of observation. Furthermore, even in the future when mass spectrometry will become the center of molecular detection and identification in the diagnosis of diseases and the study of pathological mechanisms from diseased tissues, etc., these analyzes can be performed by collecting very small amounts of biological samples. Thus, pain and burden on the patient are alleviated, and various merits are born, such as a small amount of solvent used. In this description, the most complex and organism of living organisms as a microscopic space body, and cells that are still unclear are taken up as examples of the most difficult to apply microsamples. As long as it is in a solid state, it is possible to detect almost all of the sample without any additional separation method. Further, the present invention will be described, which has a feature that can be performed while observing the minute area by observation or position information.

  In molecular analysis of cells, conventionally, a large number of cells are usually compared before and after being subjected to constant treatment, constant treatment, or constant stimulation. If we can develop a method to quickly analyze molecular dynamics while observing cells in real time and observing the behavior of one cell, we will accelerate the life sciences of the world at once. In other words, this is a dream of life science. It is also useful as a useful method for tracking molecular changes in all microscopic areas such as nanotechnology, chemistry, and material chemistry, and the realization of such a method is desired. In addition, if molecular analysis is possible with an amount of 1 picoliter or less, which is the volume of one cell, it is possible to perform molecular analysis and diagnosis with almost no pain by collecting a very small amount of blood, sweat, saliva, etc. In addition, even a very small amount of body fluid such as urine such as small animals and insects can be analyzed with 1 picoliter and can be analyzed without using a fatal amount, and its uses are infinitely wide.

We have already applied for a new method to solve the above problem.
Japanese Patent Application No. 2007-286208 Japanese Patent Application No. 2008-095092 The patent is about 1 picoliter and its volume is very small like a cell, but the tip is a nanospray tip, whose tip is a micropipette with a diameter of several micrometers such as the tip of an eyelash hair. It was only possible to capture the sample and to leave the concentration of the tip sample undiffused. However, many biological samples are viscous, and nanospraying of liquid components by applying an electric field does not occur in this state. Therefore, by further bonding an ionized solvent containing an organic solvent behind it, when the solvent passes through a very small sample at the tip of the trap, the components that elute in the solvent are discharged together as a nanospray. This was the first time in the world that it was possible to ionize components in a sample and introduce them directly into a mass spectrometer. However, after that, when applying the present invention, it was found that there was a time-consuming process unless it was mastered. That is, since sampling is in the micron range of 1 cell level, it takes time and skill to quickly bring the tip of the sampling needle there. I realized that it was necessary to make the world's first wide-ranging method easy to operate so that anyone could use it quickly anytime. Moreover, the operation can be speeded up also in the conventional method (microinjection) of injecting the component liquid into the cells. Furthermore, it is also important to further expand the target molecules and ions, and although we have applied for the basis for future medical diagnostic methods, it has become necessary to construct peripheral technologies that lead to various applications. .

The present invention addresses the following problems by using a limited space position automatic transfer system such as a robot to collect a sample such as a fragile needle at the tip when a minute sample is collected. Is continuously moved, enabling work to be performed with high efficiency.
While analyzing the cellular molecular mechanism, which has been analyzed only in aggregate until now, with one cell, including the individuality of the cell, the component is captured even to one cell or its organelle, Realize a highly sensitive, high-speed, direct, and highly reliable method that enables molecular analysis and quantification, or cells in a living tissue that is one of the cell aggregates like cancer tissue It is possible to directly extract at least one component in the formation space of the cell space, and to improve the operability to the microscopic area by realizing a means for analyzing the component as quickly and directly as possible. Provide a solution.
Moreover, cells are living creatures, and their behavior and intracellular molecular dynamics are often linked. Therefore, in order to elucidate the molecular mechanism of the life phenomenon, it is desirable that the above analysis means be performed quickly simultaneously with the observation of cell morphology. In addition, cells and cells in living tissues respond to various factors from the outside world, and in order to elucidate the mechanism of their functional expression, a method of quickly examining the relationship between factors given from the outside world and intracellular molecular fluctuations It is also necessary to provide. As a result of the discovery of the above method, behavioral analysis, molecular dynamics analysis and molecular search analysis can be performed simultaneously on at least one individual cell, providing a means to quickly and directly elucidate the molecular mechanism of life phenomena. However, the operation was cumbersome, and it was recognized that it was not easy to manually bring the tip of the component capturing device into a minute space with high reproducibility. Floating cells and eggs are gently aspirated and captured with another manipulator, and then the original manipulator equipped with a capillary capture device such as a nanospray tip is inserted under the microscope with a handcuff to insert the prescribed components. They have been picked up and injected with certain components. This was a double difficult task. In addition, while confirming the field and position of the cell to be observed, the cell is irradiated with micro light using a single optical fiber, a probe that emits near-field light at the tip is brought into contact with the cell, or a fine magnet is used. It is possible to collect intracellular components captured on magnetic beads in the micro range, insert electrodes into cells, etc., or something like a molecular capture needle that binds affinity molecules to the surface. From now on, it will be an era when various microscopic operations are required, such as insertion from the cell wall into the cell. These must be made highly efficient. In recent years, with the advancement of nanotechnology, it has become necessary to observe changes in substances in the microscopic range of 200 μm or less, and to grasp the molecular changes along with the actual state of observation. In order to provide a means for elucidating the mechanism and the like, it was indispensable to quickly and accurately move the component capture / injection device to the microscopic area in sample supplementation and component liquid injection.

Furthermore, it is currently desirable to capture components that are as painless as possible for various diseases and diagnoses. As a means of doing this, if the amount collected is extremely small, blood can be analyzed with a small sample that has been ejected or with a sample that has flowed into a fine needle by inserting it with a small diameter needle that does not hurt the earlobe. In addition, it can be diagnosed with saliva, sweat (the amount ejected from one sweat gland is about a nanoliter) and tears that are not painful at all. At that time, it would be immeasurable if it would be possible to automate the insertion of a sampling needle or holding the tip at the target location without requiring skill or effort. Furthermore, according to this technique, for example, the position of the blood vessel under the skin is determined from the near-infrared or infrared image emitted from the blood, the skin surface of the objective lens and the strongest depth of the infrared light, and the position of the blood vessel that is particularly difficult to understand for women You can also complete a system that automatically inserts the
For diagnosis of diseases, enzymes specifically present in each tissue leaked into blood are also detected. In this case, in a conventional clinical test, the enzyme activity is measured one by one by adding a substance that becomes a substrate of the enzyme and coloring it by utilizing a reaction coupled with the enzyme reaction. According to this method, it is possible to measure even a trace amount of blood, saliva, or sweat of 1 nanoliter or less from sweat glands, so that such a substrate is present in advance on the inner wall of the nanospray chip by coating, etc. I would like to be able to analyze the functions of proteins and other substances that are used in the diagnosis of deviating enzymes in the blood from the ratio of the substrate and its reactants.

In the food and drink field, alcoholic beverages such as wine, sake, beer, whiskey, and shochu, fermented foods such as soy sauce and miso, processed vegetables and fruits such as pickles, kimchi, and pickles, and dairy foods such as yogurt and cheese In the microbial cells such as yeast and lactic acid bacteria, the analysis and research at the gene level are also progressing. However, if you want to study subtle qualities such as flavor and aroma, there are limits to the analysis of these genes and proteins alone. The emergence of analytical means of intracellular low molecular dynamics of microorganisms related to fermentation such as yeast and lactic acid bacteria, and real-time component analysis in the manufacturing process such as fermentation are desired. Integration with the analysis results is desired. If a very small amount of analysis, such as a fermentation process, can be done easily, at low cost, and automated, the benefits are immeasurable.
Furthermore, a reaction system, for example, a sample that undergoes a chemical reaction such as synthesis, derivatization, or molecular bonding, can be captured at the tip of a nanospray chip and analyzed immediately to check the progress of the reaction.

  Regarding plant research, there are many unexplained events in general in various research fields such as general plant physiology and embryology compared to animal cells, and molecular mechanisms governing plant differentiation, shape, color, and fragrance. Research on the dynamics of small molecules in response to environmental responses such as gravity and light of plants is desired. In recent years, the demand for genetically modified plants has increased due to food conditions, etc., and not only ecological risk assessment but also safety assessment by genetic modification and rapid assessment of breed improvement are very important issues. Thus, the emergence of means for tracking changes in small molecule dynamics inside and outside cells is desired. We have recently succeeded in molecular analysis of a single plant cell, but fixing a plant leaf or stem in the space and inserting a nanospray chip for cell collection into it in three dimensions is possible. It is not easy and automation is desired.

  Furthermore, in general chemical manufacturing industries, etc., for example, in the manufacturing process of products that require high purity, such as organic semiconductors, organic conductors, and organic optical materials, or health assurance of food additives, etc. In manufacturing processes where important factors influence the required quality such as physicochemical performance or safety in the manufacturing process of important products, manufacturing management and quality control such as monitoring and control of such trace factors Therefore, automatic analysis of rapid molecular dynamics in the microscopic area is desired.

  However, a minute object such as a cell observed in an optical microscope is a very small cell of about 10 microns. Depending on the magnification of the objective lens, the viewing range within the field of view of the microscope is at most 1 mm or less. It is very difficult to guide the tip of a nanospray ionization capillary tip, such as a needle with a tip diameter of several microns, from the outside of the field of view, including the vertical (Z-axis) direction outside the focus of the microscope. This is the most difficult task for capturing microscopic components such as single cell component capture, which is time consuming and laborious. The higher the magnification of the objective lens, the more difficult it becomes, and the tip of the tip is mistakenly placed on the bottom of the petri dish. This is an operation with many failures such as breaking the tip. If the time and effort of this operation can be shortened, the above-mentioned new analysis method was considered to be a method used in various fields by anyone in the world.

  Therefore, this nanospray ionization capillary tube chip (hereinafter referred to as a chip) for capturing a microscopic sample such as one cell is fixed to the tip of a three-dimensional spatial position recognition moving body such as a multi-axis robot, and the chip is attached when fixed. Sometimes it recognizes and corrects the deviation of the tip of the tip on the micrometer scale due to variations in the length of the tip during manufacture or a subtle difference in the fixing point of the tip. It was thought that it would be possible to move to a designated micro-region sample position at a stretch in a predetermined movement space while maintaining a predetermined trajectory and the orientation of the tip. According to this, induction to the vicinity of the target sample in the micro area of the tip of the tip, which takes the most time and labor, can be realized with good reproducibility in a few seconds, and then the operation of adding a solvent to the component captured at the tip of the tip, The robot places the sampled sample in a rack once and releases the tip from the tip of the robot, and then the robot picks up a micro dispenser that can put the specified amount of solvent, and the solvent sampling at the tip Hold the micropipette tip for use, and after sucking a certain amount of solvent, from the rear end of the nanospray tip that has captured components such as cells, insert the plastic capillary tube like the micropipette tip thread into the nanospray. Insert into the chip and add ionization auxiliary solvent to the sampling chip, then the robot will Return it to the specified dispenser holder, release it, pick up the nanospray tip again, drop the solvent toward the tip of the nanospray tip by removing vibrations or adding centrifugal force with the tip end facing downward, and remove bubbles, etc. Then, this chip is placed on a dedicated nanospray ionization attachment of the mass spectrometer, and one cycle of the operation is completed, and a new nanospray chip is taken up from the chip rack in preparation for capturing microregion components such as the next cell. As a result, on the mass spectrometer side, molecular analysis of microscopic components such as cells by nanospray ionization starts simultaneously in parallel.

Since robots are servo controlled, hunting was predicted as a result of vibration control to maintain a certain point by servo control. It was found that the position in the vicinity of the cell does not move within a range where there is no problem with the capture, the position is maintained at the position once set, with very high reproducibility and high position accuracy. It was also found that the tip of the chip can be guided to a predetermined position near the cell with good reproducibility even after returning to a position away from the microscope by tens of centimeters. One reason may be that the chip holding portion can be manufactured in a light weight. It was surprising to find that even a robot with a manufacturer's accuracy guarantee of about 10 microns could maintain the spatial accuracy at about 1 micron if the tip was manufactured lightly. In order to achieve this, the platform was devised, and a high-rigidity frame was welded to the bottom of the platform that connects the microscope and the robot. This was also an important point for anti-vibration measures and rigidity improvement for preventing tip tip shaking. This proves that the multi-axis robot can be applied to micro operation. As a result, a series of operations can be performed automatically even if it is quite complicated.
[Patent Document 2] Although already described in detail in Japanese Patent Application No. 2008-095092, if a conventional micromanipulator is electrically operated and a tip tip measurement method is taken into account, it can be used within a small operating range (FIG. 1). However, the delivery of the chip to the mass spectrometer is long and impossible. If this conventional micromanipulator is held on a linear actuator or the like that can move linearly with high accuracy, the spatial movement distance can be increased and a simulated operation can be performed on the robot. However, detailed operations such as solvent filling are difficult unless the place of placement is very limited.
further,

  If the tip position of the nanospray tip is measured by the successful adoption of a multi-axis robot for sampling of microscopic samples accompanied by observation with this microscope, it is possible to capture the sample in the microspace targeted within the microscope, such as capturing one intracellular component. High-speed tip tip guidance to the vicinity of the target, such as at least the cells until capture, and insertion and suction from the target position to the cell etc. by simple movement with the joystick etc. succeeding to it can be performed easily and at high speed. Became. Furthermore, after aspirating the sample such as intracellular components, the solvent can be injected into the chip and then transferred to the dedicated nanospray attachment of the chip mass spectrometer.・ Automation can now be achieved.

  [Patent Document 2] One-cell capture electric micromanipulator system described in Japanese Patent Application No. 2008-095092   Quick change holder for nanospray ionized capillary tips for automation   Examples of sheathed nanospray ionized capillary tips and sheath shapes for automation   An example of a sheathed chip holding rack shown in FIG.   Nanospray ionization capillary tube tip formed by softening and expanding the upper part of the glass capillary tube at high temperature   Automatic nanospray ionization tubule tip cell guidance system with tip tip measurement monitor correction device using multi-axis robot   Automatic nanospray ionization tubule tip cell guidance system with a tip tip simple measurement monitor correction device using a multi-axis robot   Chip tip measurement system by cutting off the center of a laser   The system shown in FIG. 6 adds a function of taking out the chip from the chip holding rack and returning the chip to the original rack after capturing the cell components.   In the system of FIG. 9, a system in which a chip is further added to the nanospray ionization system of the mass spectrometer after adding a solvent to the chip.   In the system of FIG. 9, the tip holding part at the tip is a conventional micromanipulator, which makes the final fine operation easy.   The system shown in FIG. 10 is added with a system that enables suction and capture of a minute space sample such as a cell by the foot pedal operation and prevents mixing of the culture medium by pressurization.   Robotized automatic cell capture system placed in a constant temperature chamber necessary for maintaining cell growth   Cell capture experiment using robotized automatic cell capture system   Microtremor test using a joystick near the cell of a robotized automatic cell capture system   Multiple robots are installed, and the sample that moves the position of floating cells and eggs at the same observation point is lightly aspirated and captured by another manipulator, and a capillary capture device such as a nanospray ionization capillary tube is attached. An example of collecting components with a robot toward the same observation point with the original manipulator   While installing multiple robots and confirming the field and position of the cell to be observed, the cell is irradiated with micro light using a single optical fiber, or a probe that emits near-field light at the tip is brought into contact with the cell. However, an example of capturing changes in cells due to the conditions as a component change with a sampling tube at the tip of another robot   The tip of the sheathed tip shown in FIG. 3 for sample capture inside the tip nanospray ionization capillary tip can be easily introduced with a tip of the tip of the tip for solution injection by a robot, An example of providing a slope between the inner diameters of spray ionized capillary tips   Example of overall operation when this robot is a frame moving type   An example of a system that enables human blood to be collected from the arm using this robot   Example of the figure when capturing the nanoliter-level sweat that spouts from one sweat gland seen in the fingerprint area of the finger at the tip of the nanospray ionization capillary tip with this robot   Example of mass spectrum detected from sweat captured in FIG. 21 and identification molecules therein

  First, in order to automate the capture of cell components, it is necessary to enable quick replacement of the nanospray ionization capillary tube chip 1 used in large quantities at that time. In FIG. 2, a filament 338 is arranged in the nanospray ionization capillary tip 1 in order to maintain the capillary phenomenon at the tip so that nanospray is generated stably. The nanospray ionization capillary tip 1 with the metal coating applied to the outer surface is held at the center of the tip holder 340 through two O-rings 341 without air leakage, and two O-rings at the rear end of the tip holder. Thus, it is held by the robot tip attachment portion 363 of the nanospray ionization capillary tube 1 at the tip of the robot without air leakage. To replace the chip 1, pinch the chip and pull it out. This makes it possible to quickly replace the chip. Further, the nanospray ionization capillary tube tip can be used as a microcapillary without metal coating and for injection of component liquids. In the following description, the injection is not described, but it is possible.

  In order for the robot to keep the tip 1 on the robot tip attaching portion 363 one after another without air leakage, a tip coupling sheath having a circular taper inside is provided at the rear end of the tip like the sheath portion of the injection needle. can be solved. FIG. 3 shows an example of a nanospray ionization capillary tip 1 having variously shaped sheaths. Compared with the one having a circular cross section, the one having a square cross section rotates a little when the chip 1 is bitten into the chip holder when the chip 1 is held at the tip of the robot. This is because it is easy to completely bite the chip 1 without causing the tip 1 to rotate. Furthermore, the reason that some collars are attached to the sheath is to make it difficult to fall off the rack. For these sheaths, the most convenient manufacturing method would be to mold and add plastic to the nanospray tip. When the nanospray tip is made by heating and stretching the glass, a sheath may be formed on both ends before stretching, and then stretching may be performed.

  FIG. 4 shows, as an example, a rack that holds a nanospray ionized capillary tip 1 with a collared square sheath 348. In this case, a chip after capturing cells or the like, a chip waiting for solvent addition, or a chip waiting for mass analysis or after completion can be held in these six holes and placed.

  FIG. 5 shows a case where the sheath of the nanospray ionized capillary tip 1 with the filament 338 is formed by heat extrusion molding such as glass which is the same tip material. Like the addition of a plastic sheath, molecular peaks such as plastic plasticizers are not likely to appear in the mass spectrum, and during recovery, only glass and coating metal are used as raw materials, and reproducibility is excellent. The protruding portion 359 formed at the time of sheath extrusion is designed to receive rotational movement when the chip in the rack is rotated into the chip holder by rotation.

  FIG. 6 shows an example in which a micro-component capturing system such as a cell is formed by a multi-axis robot 360 using the chip 1 as described above. In this case, a 6-axis robot is used, but the robot is controlled by a small robot control unit 361. First, the tip tip position is slightly different due to the difference in biting on the holder and the tip tip length variation. Therefore, the tip position correction is performed separately from the microscope and the three-dimensional position microscope 376 (X The corrected absolute coordinate value is detected by moving the tip position to the tip point 402 displayed on the monitors 377 and 379 on the −Y plane) and 378 (XZ plane) with a fine movement device such as a joystick 405. The tip end position coordinates and the Z-axis movement encoder value of the microscope stage 373 are obtained from the microscope X-Z stage driving unit 401. For example, the focal plane which is the center of the visual field is set as the chip capture point / focal plane 403, and the guide coordinates are set. Decide, and let the robot only move. In this way, in the fine space, the focus position of the microscope and the tip position of the chip 1 operated by the robot are shared in absolute coordinates or relative coordinates, and further, the position information such as obstacles is stored in the computer in advance. By doing so, it becomes possible to control the target tool such as the chip 1 to the target fine sample with high accuracy and accuracy, including the posture such as the trajectory and orientation in the moving space. . When the tip of the chip comes near the cell, the color of the cell monitor screen 372 suddenly changes because the tip of the chip blocks light. If the absolute position between the robot and the microscope is not accurate, the chip cannot be guided with good reproducibility. Therefore, the rigidity is strengthened via the rigidity reinforcing material 400 of the mounting base as the backing of the mounting base 362 of the robotized micro area automatic sampling system. It is also important to keep it. In this manner, a microscopic sample such as a cell can be easily captured with high speed and high accuracy and good reproducibility. In addition, the vacuum suction for sucking components into the chip and the light pressurization so as not to put the culture solution or the like into the chip 1 when approaching the cells or the like in capturing will be described later with reference to FIG. .

  FIG. 7 shows a simplified tip tip position correction in FIG. 6, and the tip tip is displayed on the monitor 377 only with a microscope and a three-dimensional position microscope 376 (XY plane) separately provided separately from the microscope. This is what I did. The correction in the Z-axis direction is a cost reduction using the characteristics of the 6-axis robot, as can be seen by rotating the robot 90 degrees to show the chip position. The rest is the same as FIG.

  FIG. 8 shows another method for measuring the tip of the tip. When the tip of the tip moves to a very small and focused point by moving the X, Y, and Z axes, the passing light intensity increases. Take advantage of changing. When the vicinity of the tip crosses this small focused light path, a large change in the intensity of transmitted light occurs in a very narrow range. While moving the chip 1 with the X-axis, Y-axis, and Z-axis robots, the tip position can be determined by the change in light intensity, and the absolute coordinates at that time can be used as the tip coordinates of the chip 1. Further, the rotation of the rotation axis of the robot 363 can be corrected by the deviation from the central rotation axis of the chip 1.

  FIG. 9 shows the system shown in FIG. 6 in which the chip is further removed from the rack, and the chip is placed in the rack after the cells are captured. After the sample is collected, the chip is placed in a rack. After that, for example, the robot picks up a microdispenser that can put a specified amount of solvent, holds a micropipetting tip for solvent measurement at the tip, and keeps it constant. After suctioning a certain amount of solvent, insert a plastic capillary tube like a micropipette tip thread into the nanospray tip from the rear end of the nanospray tip that has captured components such as cells. After adding the sample to the sampling chip, the robot can return the micro-dispenser to the predetermined dispenser holder and release it.

  In addition to FIG. 9, FIG. 10 further advances the automation by causing the robot to transport the chip to the mass spectrometer. The robot once again picks up the nanospray tip 1 to which the solvent has been added, drops the solvent toward the tip of the nanospray tip by applying vibration or centrifugal force with the tip end downward, removes bubbles, etc. The chip is left in the mass spectrometer's dedicated nanospray ionization attachment, and after one cycle of operation, a new nanospray chip is picked up from the chip rack in preparation for the capture of the next cell and other microregion components. As a result, on the mass spectrometer side, molecular analysis of microscopic components such as cells by nanospray ionization starts simultaneously in parallel.

  FIG. 11 shows a conventional small-sized XYZ microphone manipulator that can change the fine movement operation for fine cell movement, such as the last cell capture, in the micro sample collection of cells or the like by only the robot. The case where is attached to the tip of the robot is shown. Although the robot tip must be heavy and the weight of the robot itself does not cause hunting, fine movement is smoother.

  FIG. 12 is a diagram of FIG. 11 and the previous drawings, in order to capture aspiration of a microscopic sample such as a cell with the nanospray tip 1, rather than using a hand-held suction operation, a hand that tends to be blocked is used. However, the piston 436 is moved by the operation of the pedal 437 by the foot, and the vacuum solution for sucking the component into the chip at the time of capture or the culture solution or the like is approached to the cell or the like through the tube. It allows light pressurization so as not to enter. It is also possible to put a liquid into the tube in order to improve the operating characteristics of suction and pressurization. This operation can also be automated by electric drive, but the operation is not smooth and appropriate.

  FIG. 13 is for maintaining the above system at around 36 degrees Celsius, which is an appropriate temperature for cell observation. In particular, this is a carbon dioxide concentration maintenance and constant temperature chamber essential for culturing cells and observing them for a long time. The front is not made of double glass but is made of a single piece of organic glass, and is provided with a microscope observation window 453, a microscope operation window 454, a chip take-out window 455, and the like. You can also. In that case, a large transparent door 457 is provided to maintain the mass spectrometer and its ionization attachment.

  FIG. 14 is a photograph in which cell insertion is actually performed using a 6-axis small robot.

  FIG. 15 shows that the fine movement operation is performed by the joystick operation at the final stage of the intracellular insertion in FIG. Both showed performance exceeding expectations.

  FIG. 16 shows an example in which a plurality of 6-axis small robots are installed. Unlike the sticking cells such as floating cells and eggs in the left robot, the tip diameter is the target for the sample whose position moves. It is a small tube that is close to the size of the sample and has rounded corners so that it will not be damaged, and it is captured by weakly aspirating, and the capture position can be determined from the coordinate axis value of the robot. This is a system that can guide the tip of the chip. When a plurality of robots are used in this way, an operation that takes advantage of the characteristics of a robot that can share absolute coordinates is further possible, and high accuracy and high efficiency of work can be achieved.

  FIG. 17 shows a probe that places a plurality of robots, confirms the field and position of the cell to be observed, irradiates the cell with micro light using a single optical fiber, and emits near-field light at the tip. This is an example in which a change in cells due to the conditions is captured by a sampling tube located at the tip of another robot while being in contact with the sample. For measurement samples (in this case, cells), it is possible to collect intracellular components on magnetic beads with other fine magnets in the micro range, and the electrodes are pressed microscopically or inserted into cells. In the future, various micro-range operations will be possible at the same time, such as inserting a molecule-capturing needle with affinity molecules attached to the surface into the cell through the cell wall.

  18 is a tip of the sheathed tip shown in FIG. 3 for sample capture, and a tip 482 like a tip of a tip for solution injection can be easily introduced from the rear end of the tip by a robot. In this example, a slope 480 is provided between the coupling portion of the sheath and the inner diameter of the nanospray ionization capillary tube tip. After the robot captures a sample component such as a cell with the tip, the tip is once placed in the rack. In addition, by making use of such a sheath shape, a solvent can be easily added to the inside of the capillary using the solution addition capillary tip 481 from the rear end of the tip. Until now, this important solvent addition work has been difficult and difficult, and has been a hindrance to efficiency.

  FIG. 19 shows an example of the overall operation when the type of the robot is a frame moving type. This type of robot that moves using the frame as the movement axis 501 is low in cost, but has many restrictions on the movement space, and the use is narrower than the above six-axis rotation type robot. There are other arm types and various robots, but the basics of the operation remain the same.

  FIG. 20 shows an example of a system in which human blood can be collected from an arm 510 by this robot. If a camera 371 having sensitivity in the near infrared region or the infrared region is used for the camera, the position of the blood vessel 511 having a high temperature can be easily visualized. If the size and position of the blood vessel can be confirmed from the focal plane, the human arm 510 is fixed with a biological sample collection point determination plate 514 at the blood collection position, and a biological sample collection needle 515 such as an injection needle is inserted there. To do. The passage through the blood vessel wall at that time is sensed by a touch sensor 518 at the time of collection provided at the base of the needle, and the robot is stopped at the place where one side of the blood vessel wall is inserted to enter blood collection. A signal from the sensor is amplified by a preamplifier 521 via a sensor signal line 520 and sent to a robot drive computer.

  FIG. 21 is an example of a diagram in which nanoliter-level sweat sprayed from one sweat gland seen in the fingerprint region of a finger is captured by the tip of the nanospray ionization capillary tube with this robot. Sweat 530 spouted from one sweat gland in the fingerprint region of the finger was captured by the nanospray ionization chip 515, and a solvent was added to this from the rear end, and a molecular peak was directly detected by nanospray ionization mass spectrometry. . FIG. 21 shows a mass spectrum that was successfully detected. In the mass spectrum obtained from one drop of sweat, there are 130 molecular peaks specific to sweat. Among them, three examples of molecular peaks identified with amino acids and their molecular structures are described. Successful detection of ingested caffeine and nicotine, and the drug molecules that are being taken, this success can be done in a variety of ways, such as in the future, not with blood, but with less painful samples on the patient. Showing sex.

Cells that move purposefully and respond to external factors are often linked to their behavior and intracellular molecular dynamics. The mechanism can be elucidated quickly. The present invention provides a means for quickly and directly elucidating both a life phenomenon and its molecular mechanism on a micro scale up to one cell, and its application is enormous. Above all, elucidation of the molecular mechanism of the disease by kinetics and molecular comparison of diseased cells and normal cells is accelerated. If the intracellular molecular mechanism is known, drug discovery, diagnostic methods, and therapeutic methods using the molecule or mechanism will be developed, and if a new molecule is found, it will be applied to new drugs or life science reagents. Application is possible.
In addition, speeding up the elucidation of various molecular mechanisms in cells and in living tissues has led to faster discovery of new molecules, including drug candidate molecules, and discovery of new life phenomena. Can be greatly expanded and accelerated. For example, various analyzes such as rapid search for cell differentiation factors of universal cells such as regenerative medicine, cell differentiation control, discovery and control methods of cell growth factors, molecular diagnosis and search for cell types such as cancer cells, and cell type identification Will be deployed at high speed.

  It is possible to clarify the dynamics of small molecules in cells in relation to many genes that are currently being comprehensively analyzed for various diseases and proteins that are their expression products. Clarification is possible, and an integrated understanding of life phenomena can greatly contribute to the enhancement of human life and health, and can create various incidental businesses.

Further, in the food field, it is possible to provide a direct and rapid means for analyzing low molecular dynamics such as intracellular and fermented liquid that can study delicate qualities such as flavor and flavor.
Furthermore, in the general chemical manufacturing industry, for example, elucidation of the nano-range molecular mechanism of nanotechnology, production management of products that require high purity such as organic semiconductors, organic conductors, and organic optical materials, or food addition In the manufacturing process of products that require quality assurance for health, such as by-products, in the manufacturing process where trace amounts of by-products adversely affect the required quality such as physicochemical performance or safety, such by-products Molecular detection and analysis in the micro range, such as monitoring, manufacturing control, quality control, etc. becomes possible.

1 Nanospray ionization capillary tip 2 Mass spectrometer 3 Sample solution and added ionization solvent 4 High voltage (in this case, positive mode, tip side is +)
5 Nanospray 6 Metal coating part on the surface of tubule 8 Cell 9 Microscope stage 10 Captured cell solution 11 Objective lens 12 Video camera 13 Suction operation 15 Petri dish 16 Cell culture solution 17 Monitor 35 Drive cell insertion nanospray tubule tip to cell position XYZ drive stage of the holder to perform (In this figure, there are also electric type and manual gear coarse / fine movement type)
36 Axial actuator of holder that drives the cell insertion nanospray capillary tip to the cell position (In this figure, there is also an electric type, manual hydraulic or hydraulic piston drive type)
37 Cell Insertion Nanospray Capillary Tip Driven by Axial Actuator that Drives Cell Insertion Nanospray Capillary Tip to Cell Position 38 Cell Insertion Nanospray Capillary Tip Holder Drive Controller 39 Cell Insertion Nanospray Capillary Tip Holder Drive Terminal (joystick in this figure)
40 Tube for cell component suction drive 41 Piston for cell component suction (can be replaced by syringe)
42 Piston drive device 338 for cell component suction Nano-spray tube attached filament 340 Nanospray suction holder 341 Sealed O-ring 342 during nanospray suction Holder attachment O-ring 343 Nanospray suction holder body 344 Nanospray ionization tubule with suction sheath Tip (Surface metal coating can be coated only with a thin tube or with a sheath)
345 Concentric sheath (manufactured by injection molding, etc., the joint with the suction part is a round taper)
346 Square sheath (manufactured by injection molding, etc., the joint with the suction part is a round taper)
347 Square sheath with a partial collar (manufactured by injection molding, etc., the joint with the suction part is a round taper)
348 Square sheath with surrounding collar (manufactured by injection molding etc., the joint with the suction part is a round taper)
349 Nanospray ionized capillary tip storage rack 350 with sheath Hole for storing nanospray ionized capillary tip with sheath (square)
352 Heating filament 353 Nanospray ionization capillary tip opening rear end spreading die 354 Nanospray ionization capillary tip opening rear end pushing die 355 Integrated nanospray ionization with the finished rear end sheath structure Capillary tip (metal coating can be done before or after spreading, but better later)
359 Positioning projection 360 formed by changing the shape of the die on an integral nanospray ionization capillary tube having a rear end sheath structure A small robot (a 6-axis control robot is shown as an example)
361 Small robot controller 362 Robotized micro area automatic sampling system mounting base 363 Nanospray ionization capillary tip robot mounting section 370 Automatic micro area sampling microscope system 371 Microscopic analysis video camera and objective lens 372 371 Fig. 373 Microscopic stage 374 Microscope XY stage drive unit 375 Cell culture dish 376 Video camera with objective lens for measuring tip of nanospray ionization capillary tube (XY plane)
377 Tip of tip captured by 376 Figure 378 Video camera with objective lens for tip measurement of nanospray ionized capillary tip (XZ plane)
Tip tip 400 captured by 379 378 Stiff stiffener 401 for robotized microscopic automatic sampling system mounting stand Microscope X-Z stage drive 402 Tip of nanospray ionization capillary tip 403 Tip capture point / focal plane 404 Microscope focal plane Z-axis fluctuation encoding communication line 405 Robot fine attachment (in this case, joystick)
410 Light source for tip detection (in this case, laser, etc.)
411 Round hole slit for light source 412 Light source angle changing mirror 413 Condensing lens 414 Condensing point / pinhole slit 415 Photodetector 420 Sample captured in chip Nanospray ionization device 421 Nanospray ionization capillary tube fixing / voltage application unit 425 Mass analysis In-chip capture sample nanospray ionization device 430 as a gauge attachment XYZ micromanipulator drive (various types such as water pressure, hydraulic pressure, and electric power)
431 X-Y-Z micromanipulator drive tube (hydraulic, hydraulic) or wire (electric)
432 XYZ micromanipulator Rotating handle for fine driving 435 Sample suction tube 436 Sample suction piston syringe 437 Pedal 438 Pedal rotation shaft 439 Pedal swing arm 440 Pedal sample suction pressure controller 450 For cell capture Or a constant temperature chamber 451 for cell capture molecular analysis integrated system A constant temperature chamber base 452 for cell capture Double glass or double organic glass 453 Microscopic observation window 454 Microscope operation window 455 Sample capture chip extraction window 456 Heat source (oil heater, etc.)
457 Mass spectrometer operation door 460 Floating micro sample 461 such as floating cell or egg Cell holding robot 462 Cell holding pipette 463 Piston syringe 470 for holding micro sample such as cells, suction and injection 470 Optical fiber 471 Light source 472 Optical fiber holding rod 473 Irradiated light 480 Solution addition capillary introduction slope 481 Solution addition capillary tip 482 Solution addition capillary tip tip 483 Solution added to the capillary 500 Frame moving robot frame support 501 Robot movement horizontal frame 505 Robot rotation unit 506 Robot frame moving unit 510 Human arm (an example of a living body)
511 Blood vessel 514 Biological sample collection point determination plate 515 Biological sample collection needle 516 Biological sample collection suction piston syringe 517 Biological sample collection piston drive unit 518 Touch sensor 520 at the time of collection Sensor signal line 521 Sensor signal preamplifier 530 Sweat gland in the fingerprint region of the finger Sweat erupted from one 540 Mass spectrum obtained from one drop of sweat and identified molecule

Claims (7)

In suction capture from a micro sample such as a cell or injection into the sample, the absolute space coordinates or relative space coordinates of the tip of the sample suction capillary or the component liquid injection capillary held at the tip of the robot and the observation of the sample The absolute space coordinate or relative space coordinate of the suction point or injection point on the observation focal plane in a microscope for use is defined, and the capillary tube is automatically moved to a predetermined position according to the calculated displacement amount of the robot arm. Apparatus and system for guiding the trajectory of the space from the storage position of the thin tube to the minute sample area by the robot while maintaining the posture such as the direction of the predetermined thin tube 2. The robot according to claim 1, further comprising one or more robots having the same function, wherein the plurality of robots are fixed with a target micro sample such as a cell that is moved and whose position is not determined in the space. A device that can perform secondary operations at the same time by applying light, applying a magnetic field, or bringing a rod-shaped micro-equipment with a tip that captures molecules into the sampled space. And system The apparatus according to claim 1, wherein after the sample suction and capture, the solvent is automatically added to the capillary by the same robot or another robot, and then the chip is mounted on the nanospray ionization attachment of the mass spectrometer. And system 4. The robot according to claim 1, wherein the robot moves the capillary tube close to the target sample, and the subsequent operation uses an interface such as a joystick to finely drive the robot, or hydraulic or water pressure or An apparatus and system that holds an electric micromanipulator and allows finer operation from a predetermined position 6. The apparatus and system according to claim 1, wherein the suction or pressurization of the sample suction capillary tube or the component liquid injection chip is controlled by the movement of a piston operated by a foot through the tube. Claims 1, 2, 3, and 4 wherein the apparatus or system is placed in a chamber maintained at a carbon dioxide concentration or temperature at which cells grow, and the atmosphere from the outside is minimized through sleeves formed in the chamber window. Device and system that can be operated without changing Nanospray ionization chip with a sheath to easily connect with a robot holder and cylinder with piston so as not to leak air, and the above coupling of the sheath so that a solvent introduction capillary can easily enter from the rear end. Nanospray ionization tip with sheath that connects the inner surface of the holding part and the inner diameter of the tip with a smooth gradient

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