JP4438323B2 - Liquid dispensing device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a liquid dispensing apparatus for dispensing a sample or a reagent in an analyzer used in fields such as chemistry, industry, clinical practice, and biotechnology.
The liquid dispensing apparatus can be used, for example, as an apparatus for dispensing a liquid on a membrane in order to use a substance developed and solidified on a membrane as a sample such as mass spectrometry.
[0002]
[Prior art]
For the purpose of analyzing the mass of target molecules, a laser desorption ionization mass spectrometry method has been carried out in which a sample placed on a sample plate mounted on a mass spectrometer is ionized and analyzed by irradiating the sample with a laser beam. ing. When preparing a sample by placing it on a sample plate, there are a method using a matrix and a method not using it.
[0003]
A method in which a method using a matrix is combined with a time-of-flight mass spectrometer is called a MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight) mass spectrometry method. In MALDI-TOF, a measurement sample is dropped onto a metal sample plate together with a matrix solution, and measurement is performed after drying.
[0004]
On the other hand, as a target sample, biomolecules are separated by electrophoresis, etc., transferred to a membrane and solidified, and the membrane is solidified on the membrane using a micro-dispensing technique using a piezo element. A method of performing mass spectrometry using reaction products generated by performing various reactions is proposed (see Patent Document 1).
[0005]
There is an immunoblot method as a method for detecting a substance that is spread on a membrane and is immobilized. In general, immunoblotting is also called Western blotting, in which a protein sample is developed by electrophoresis, solidified on a membrane, and then reacted with a specific antibody against the target substance as a probe to detect its presence. is there.
[0006]
[Patent Document 1]
International Publication No. WO 98/47006 Pamphlet [0007]
[Problems to be solved by the invention]
After a reagent or sample is dispensed to a sample plate or an object with a membrane fixed on the sample plate by a dispensing mechanism such as a piezo element, the sample plate is separated from another device, such as a mass spectrometry system or other analytical device. Alternatively, transfer to a pre-processing device to perform analysis and next processing.
It is first necessary to accurately dispense reagents and samples only to the spots developed on the sample plate and membrane.
[0008]
It is preferable that the dispensing element is clogged by the dispensing mechanism, or that the dispensing element (referred to as a dispensing element) can be replaced in order to dispense another reagent or sample. In that case, when a dispensing element is newly installed or replaced, it may shift from a predetermined position.
Therefore, an object of the present invention is to enable an accurate dispensing operation even when a dispensing element is newly installed or replaced.
[0009]
[Means for Solving the Problems]
The dispensing device of the present invention includes a dispensing mechanism in which a dispensing element for dropping a sample or reagent is detachable, an image reading device for reading a lower image, and an object to which the sample or reagent is dispensed. A movable table supported on the upper surface and moved in a horizontal plane to position the object at least at a dispensing position below the dispensing element and an image reading position below the image reading device; and A dispensing position is detected based on an image read by the image reading device when dispensing is performed at a predetermined position on the movable table, and dispensing is performed based on a reference base point on the movable table that is read simultaneously. And a calibration unit for calibrating the injection position.
[0010]
The dispensing element of the dispensing mechanism is mounted at a predetermined position. To ensure that the dispensing position of the mounted dispensing element is always the same position, the dispensing element mounting part is advanced. High machining accuracy is required. In addition, there are machine differences in individual dispensing elements. For this reason, if an attempt is made to adjust the mounting of the dispensing element so that the dispensing position becomes a predetermined position, a high degree of machining accuracy and a skilled adjustment capability are required. However, in the present invention, when a dispensing element is mounted, the dispensing position by the dispensing element is detected, and calibration for obtaining a deviation from the reference dispensing position is performed, thereby mounting or replacing the dispensing element. The adjustment work associated with is simplified.
The present invention also includes one provided with a plurality of dispensing elements. In that case, the calibration unit calibrates each dispensing element.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
FIG. 1 schematically shows an embodiment of the liquid dispensing apparatus of the present invention.
Reference numeral 4 denotes a print head which is a dispensing mechanism, and four dispensing elements 10-1 to 10-4 each including a piezo element for dispensing a reagent and the like are mounted in a line. The positions of the dispensing elements 10-1 to 10-4 are fixed. In order to control the dispensing amount from the dispensing elements 10-1 to 10-4, a pressure control unit 7 for adjusting the pressure applied to the dispensing elements 10-1 to 10-4 is attached to the front surface of the apparatus main body. The four knobs 8 for adjusting the pressure in the dispensing elements 10-1 to 10-4 are arranged corresponding to the dispensing elements 10-1 to 10-4.
[0012]
A scanner 6 is arranged as an image reading device for taking a sample plate to be dispensed as an image together with a table. The position of the scanner 6 is also fixed.
[0013]
The table 2 is a movable table that can move in a horizontal plane. On the table 2, a sample plate 50 is placed at a predetermined position, as will be described later with reference to FIG. The table 2 moves in a plane, the specified position of the sample plate 50 is positioned below the dispensing elements 10-1 to 10-4 at the time of dispensing, and the portion to be imaged on the table 2 is taken at the time of taking an image. Position below the scanner 6.
[0014]
When dispensing from the dispensing elements 10-1 to 10-4, a CCD camera 5 is disposed as an imaging device to capture an image of the tip and monitor the state of dispensing. The CCD camera 5 is mounted so as to capture an image of the tip of the dispensing elements 10-1 to 10-4 from obliquely above in order to reduce the installation space. Dispensing elements 10-1 to 10-4 dispense different liquids. Dispensing elements 10-1 to 10-4 having predetermined dispensing positions such as sample plate 50 positioned downward are dispensed. Perform note operation. When the dispensing elements 10-1 to 10-4 performing the dispensing operation are changed, the camera 5 is arranged so as to capture images of the dispensing elements 10-1 to 10-4 performing the operation. It is attached so that it can move parallel to the array of 1-10-4.
[0015]
The table 2, the print head 4, the camera 5 and the scanner 6 are housed in a case made up of a main body 9a and a lid 9b, and the case lid 9b can be opened at any time, for example, when the sample plate 50 is replaced. ing.
[0016]
FIG. 2 shows a sample plate and the like arranged on the table 2. Two sample plates 50 can be fixedly placed at predetermined positions. A membrane 52 is affixed on the sample plate 50, and a reagent and a sample are dispensed by this dispensing apparatus. Samples immobilized on the membrane 52 are, for example, proteins, peptides, sugars, lipids, nucleic acids, and the like separated and developed in a one-dimensional direction by SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis or other chromatography. A molecule or a mixture thereof.
[0017]
The sample can be immobilized on the membrane 52 by spreading the sample on a gel or other electrophoresis medium and then transferring it to the membrane 52.
Examples of the material of the membrane used for the solid phase include PVDF (polyvinylidene difluoride), nitrocellulose, nylon (registered trademark), and derivatives thereof.
[0018]
In order to detect the substance in the spot which is developed on the membrane 52 and solidified, the digestive fluid and the extract from the dispensing elements 10-1 to 10-4 are applied to the sample components solidified on the membrane 52. Can be dispensed.
Moreover, the substance used as the probe couple | bonded with an object molecule | numerator can also be dispensed. As such a probe, an antibody is used when the target molecule is an antigen. In general, a biological substance that specifically reacts with a target molecule can be used. Also, several antibodies and biological materials can be used in combination.
It is preferable that the reagent is dispensed only in the area where the spot on the membrane 52 exists. Thereby, waste of reagents can be eliminated.
[0019]
In order to enable optical detection of a target substance, a primary reagent including a probe that specifically reacts with the target substance as a reagent to be dispensed, and a secondary reagent that develops the target substance after reacting with the probe Can be used. In that case, the primary reagent is first dispensed from any of the dispensing elements 10-1 to 10-4, and then from the other of the dispensing elements 10-1 to 10-4 on the area where the primary reagent has been dispensed. Dispense secondary reagent. As such a secondary reagent, a coloring reagent or a fluorescent reagent can be used.
[0020]
In addition, after reacting the probe with the target molecule and detecting it by an appropriate means, in addition to dispensing a coloring reagent or fluorescent reagent, reacting metal ions or combining these methods be able to. As such a method, there are a method using a colloidal gold labeled antibody and a method of introducing a protein having affinity for metal ions into a fluorescent reaction using Ni ²⁺ chelate enzyme or the like.
[0021]
Since a plurality of dispensing elements 10-1 to 10-4 are provided, by moving the dispensing position on the sample plate 50 by the table 2, the dispensing elements 10-1 to 10-4 are changed to a plurality. Can be dispensed.
[0022]
Each sample plate 50 is provided with a plurality of marks b. These marks b are reference points that serve as a reference when creating the dispensing position on the membrane 52 affixed on the sample plate 50 as information. In this example, reference points b are provided at four corners of a substantially rectangular shape, and these reference points b are captured by the scanner 6 together with the image of the membrane 52. One corner of the sample plate 50 is cut off to indicate the orientation of the sample plate 50.
[0023]
An area 54 provided on the table 2 is an area provided as a test print unit for performing a dispensing test using the dispensing elements 10-1 to 10-4. A filter paper is also attached there, and the dispensing state at the time of test printing can be confirmed by the CCD camera 5.
[0024]
A region 56 provided on the table 2 is a maintenance unit for the dispensing elements 10-1 to 10-4, and a sponge 57 is provided. When liquid or dirt adheres to the tip of the dispensing elements 10-1 to 10-4, the maintenance unit is moved under the dispensing elements 10-1 to 10-4, and the dispensing element is moved by the sponge 57. The liquid and dirt attached to the tips of 10-1 to 10-4 are wiped off.
[0025]
Further, a mark a is provided on the surface of the table 2 as a base point serving as a reference for the position of the sample plate 50 disposed on the table 2. The mark a serves as a reference when the dispensing position is taken out as information, and serves as a reference for matching the position on the image acquired by the scanner 6 with the movement of the table 2. Each dispensing element 10-1 to 10-4 in the head 4 has the same structure.
[0026]
An example of the liquid dispensing apparatus used for the micro-dispensing method is the one using the piezo element described above. In such a liquid dispensing apparatus, a reagent filled in a space connected to the discharge unit at the tip is pressed by a drive unit including a piezo element, thereby discharging a droplet from the discharge unit. In this case, the parameter of the control signal may include at least one of the magnitude of the applied voltage to the piezo element, the rising time of the applied voltage, the applying time, and the falling time of the applied voltage.
[0027]
As a liquid dispensing apparatus similar to this, an apparatus provided with a liquid ejection element used in an ink jet type liquid ejection apparatus can be used.
[0028]
As another example of the liquid dispensing apparatus used in the microdispensing system, a dispensing system apparatus using a syringe pump can also be used. In that case, the parameter of the control signal may include at least one of the stroke, speed, and acceleration of the plunger of the syringe pump.
[0029]
An example of a dispensing mechanism provided with a piezo chip 10 incorporating a piezo element as a dispensing element will be described in detail with reference to FIG.
A piezo chip 10 as a liquid discharge unit having a discharge port at the lower end and a hollow needle 16 at the upper end is detachably attached to a chip holding unit 31 of the liquid dispensing device by a screw 29. The piezo chip 10 can be removed by loosening the screw 29 and can be fixed by tightening the screw 29. When the piezo chip 10 is attached or detached, the dispensing position shifts. Therefore, as described later, in this embodiment, the dispensing position is calibrated with reference to the base point a on the table 2.
[0030]
Dispensing liquid container 20 is disposable, and has an opening at the lower end and the upper end, and the lower end opening is closed by lid 15 to store the dispensing liquid therein. The lid 15 at the lower end opening is an elastic septum, and is constituted by an elastic member that can be penetrated by the hollow needle 16 of the piezo chip 10 and that the through hole can be closed by elasticity when the penetrated hollow needle 16 is pulled out. . The elastic member constituting the lid 15 is, for example, rubber.
[0031]
The air introduction head 30 can be attached from the upper part of the container 20 in a state in which the container 20 is mounted on the chip 10 and mounted through the hollow needle 16 through the lid 15 at the lower end opening. The air introduction head 30 adjusts the pressure in the container 20 and adjusts the amount of liquid discharged from the chip 10.
[0032]
A seal member 32 is provided at the tip of the air introduction head 30. The seal member 32 is an O-ring, for example. By pressing the air introduction head 30 against the container 20, the sealing member 32 comes into contact with the inner surface of the opening of the container 20 to seal the opening of the container 20 while keeping the air tight, and pressure is applied to the air introduction head 30. The pressure in the container 20 can be controlled by the air sent from the control mechanism.
[0033]
An arm mechanism is provided for detachably mounting the air introduction head 30. The arm mechanism includes an arm 33 and a lock 36, and the air introduction head 30 is held at one end of the arm 33. The base end portion of the arm 33 is rotatably supported by the liquid dispensing apparatus main body by a pin 34. The lock 36 is rotatably supported by the arm 33 by a pin 38, and a flange 40 is provided at the base end portion. The ridge 40 engages with a convex portion 35 fixed to the liquid dispensing apparatus main body, and can be locked in a state where the container 20 is mounted on the chip 10.
[0034]
The air introduction head 30 is slidably inserted into a hole formed in the arm 33 and is urged by the spring 41 so as to push the air introduction head 30 toward the container 20. The base end portion of the air introduction head 30 protrudes from the arm 33, and the flange 45 is provided at that portion, so that the air introduction head 30 is prevented from coming out of the arm 33. A base end portion of the air introduction head 30 is connected to a pressure control mechanism via a pipe 44.
[0035]
A coil spring 42 in a compressed state is inserted between the arm 33 and the lock 36 in order to urge the lock 36 in the direction in which the flange 40 engages with the projection 35 around the pin 38.
A spring 43 is placed between the arm 33 and the liquid dispensing apparatus main body, and the spring 43 biases the arm 33 to be pulled in the opening direction (clockwise direction in the figure).
[0036]
In the mechanism of FIG. 3, when the container 20 is mounted, the container 20 is placed on the chip 10, pushed downward, penetrates the septum 15 with the hollow needle 16, and the hollow needle 16 is immersed in the solution in the container 20. Since the air introduction head 30 is held by the arm 33, the air introduction head 30 is attached to the opening of the container 20 by rotating the arm 33 counterclockwise in the figure, and the container 20 and the air introduction head 30 are thus mounted. Are connected to each other by the seal member 32 in an airtight manner. At this time, the collar 40 is engaged with the convex portion 35 to lock the arm 33 and prevent the arm 33 from rotating clockwise so as to open. Since the air introduction head 30 is biased so as to be pressed in the direction of the container 20 by the spring 41, the airtight connection between the container 20 and the head 30 is maintained with the arm 33 locked.
In this state, the liquid can be discharged from the chip 10.
[0037]
When removing the container 20, the lock 36 is pushed in the direction of the arm 33. The lock 36 pivots clockwise around the pin 38 in the figure, the engagement between the collar 40 and the projection 35 is released, and the arm 33 pivots clockwise in the figure due to the force of the spring 43, and the air introduction head 30 leaves the container 20.
[0038]
In this state, the air introduction head 30 rotates around the pin 34 together with the arm mechanism, so that the space between the air introduction head 30 and the container 20 opens widely, and the sealing material 32 of the air introduction head 30 and the cleaning around the container 20 are cleaned. This makes it easier to maintain.
[0039]
Further, if the air introduction head 30 can be attached to and detached from the container 20 using the arm mechanism in this way, the attachment and detachment of the container 20 and the attachment and detachment of the air introduction head 30 to the container 20 can be easily performed. It becomes like this.
[0040]
Returning to FIG. 1, the CCD camera 5 will be described.
In the field of analyzers that often use a liquid phase regardless of samples and reagents, attempts have been made to reduce the amount of solution used for analysis. This not only saves valuable sample waste and reduces the amount of expensive reagents used, but in biochemical reactions between solutions, the smaller the amount of solution, the shorter the reaction time. Therefore, it is an effective method for increasing the processing efficiency of the experiment.
[0041]
In order to carry out the reaction with a small amount of solution, a dispensing device for dispensing a sample or reagent in a minute amount is necessary. As a method for dispensing a small amount of liquid, there are various methods such as a method using a piezoelectric element such as the piezo element of the embodiment, a method by opening and closing a valve, a method using bubbles generated by locally heating a solution, etc. Has been put to practical use.
[0042]
When dispensing a small amount of liquid to a target position, delicate control of various parameters is required, such as how to apply a voltage to the element if it is a piezoelectric element, and opening and closing time if a valve is used. In order to optimize these parameters, and when dispensing to many locations, the dispensing time also becomes long, so the shape of the droplet to be dispensed is monitored and the environment where the dispenser is located In order to cope with the change and the change over time of the piezoelectric element, it is preferable to capture and monitor the image of the droplet formed on the tip of the dispensing element with an imaging device. The CCD camera 5 is an example of such an imaging device.
[0043]
By disposing the CCD camera 5 for monitoring the dispensing state at an angle with respect to the horizontal direction, the CCD camera 5 can be installed within the moving range of the movable table 2 without interfering with the movable table 2. And the dispenser becomes smaller.
A light source may be arranged at a position opposite to the CCD camera 5 across the tip of the dispensing element. In this case, the light source reflects the light emitted from the surface of the sample plate 50 as a target and dispenses. It is directed in the direction of incidence on the CCD camera 5 via the element tip. By providing such a light source, when monitoring a sample or reagent droplet formed at the tip of the dispensing element, it becomes possible to capture the image of the droplet with transmitted light, resulting in a clearer image. An image can be obtained and an accurate monitor can be performed. Further, by turning on the light source in synchronization with the dispensing timing, it is possible to capture the droplet as a still image.
[0044]
The CCD camera 5 can also be set so as to capture an image of the surface of the sample plate below the dispensing element as well as an image of the tip of the dispensing element. In that case, the state of the surface of the sample plate can be monitored together with the monitoring of the tip of the dispensing element, and more information can be obtained. For example, it becomes possible to confirm whether or not the sample or reagent is accurately dispensed at the target position. For example, when the object to which the sample or reagent is dispensed is a membrane, the membrane before and after dispensing It is also possible to observe the state of the film and to observe the temporal change of the film state during the reaction.
[0045]
As an application of this dispensing apparatus, for example, one that dispenses a reagent to a solid phase such as a PVDF (polyvinylidene difluoride) membrane can be cited. A spot developed by chromatography is transferred to the PVDF membrane, and a reagent is dispensed to develop the color of the spot. As such a solid phase, nitrocellulose, nylon (registered trademark), or the like can be used in addition to the PVDF membrane.
[0046]
FIG. 4 shows the function of the apparatus of FIG. 1 as a block diagram.
Reference numeral 60 denotes a monitor unit that displays an image read by the image reading device 6 such as a scanner. The dispensing position designating unit 62 is for designating the dispensing position on the object 50 based on the image of the object 50 such as a membrane displayed on the monitor unit 60. The dispensing control unit 64 performs relative positioning of the object and the dispensing element so that the dispensing position on the object designated by the dispensing position designating unit 62 is below the dispensing element of the dispensing element 10. The dispensing operation by the dispensing element 10 is controlled. When the dispensing element 10 has a piezo element, the dispensing control unit 64 adjusts the pressure in the dispensing element 10 by the pressure control unit 7, and the voltage to the piezo element by the dispensing control unit 66. The liquid is discharged from the piezoelectric element by controlling the application.
[0047]
The dispensing position information creation unit 68 creates dispensing position information related to the dispensing position on the object 50 that has been designated and dispensed by the dispensing position designation unit 62. The dispensing position information creation unit 68 can output the created dispensing position information to the outside.
[0048]
As shown in FIG. 2, the object 50 is provided with a plurality of reference points b that serve as reference positions within the object so that the image reading device 6 reads the image of the reference point b together with the image of the object 50. To. As a result, the analysis position information creation unit 68 can create the position on the object 50 designated by the analysis position designation unit 62 based on the plurality of reference points b. Even when mounted on the table 2, if an image is acquired by the scanner 6, the positioning can be performed accurately based on the reference point b. Further, even when the object 50 is moved to an analyzer or the like, it is possible to accurately position the dispensing position in the object 50 from the dispensing position information based on the reference point b. Become.
[0049]
The table 2 that supports the object is driven by the table driving mechanism 65 and moves in a plane, and is positioned at a predetermined position designated by the dispensing control unit 64. A plurality of base points a serving as a reference are provided on the table 2 so that the image reading device 6 reads the image of the base point a together with the image of the object 50, and the analysis position information creation unit 68 includes the object 50. The upper position can be created based on a plurality of base points a. Thereby, the dispensing position information in the object 50 can be accurately determined based on the base point a.
[0050]
The dispensing element 10 for dropping the sample or reagent is detachable and includes a calibration unit 72 for calibrating the dispensing position of the dispensing element 10. The calibration unit 72 detects the dispensing position based on the image read by the image reading device 6 when the dispensing element 10 dispenses a predetermined position on the table 2, and simultaneously reads the reference on the table. The dispensing position is calibrated based on the base point. Dispensing for calibration is performed at a plurality of predetermined positions as indicated by three points by the membrane 53 that develops color when, for example, the reagent on the table 2 shown in FIG. 2 is dispensed. This calibration is performed each time the dispensing element is mounted.
[0051]
【The invention's effect】
In the liquid dispensing apparatus of the present invention, the dispensing element of the dispensing mechanism is detachable, and the image read by the image reading device when the dispensing element dispenses to a predetermined position on the movable table. The dispensing position is detected based on this, and the dispensing position is calibrated based on the reference base point on the movable table that is read at the same time, making it easy to make adjustments when installing or replacing dispensing elements. It will be something.
[Brief description of the drawings]
FIG. 1 is a perspective view schematically showing an embodiment.
FIG. 2 is a plan view showing an upper surface of a table on which a sample plate and the like are arranged in the same embodiment.
FIG. 3 is a cross-sectional view showing a dispensing mechanism in the same embodiment.
FIG. 4 is a block diagram showing the function of the embodiment.
[Explanation of symbols]
2 Table 4 Print head 5 CCD camera 6 Image reading device (scanner)
7 Pressure controller 10, 10-1 to 10-4 Dispensing element (piezo chip)
16 Hollow needle 20 Dispensing container 29 Screw 30 Air introduction head 31 Chip holding part 32 Seal member 33 Arm 36 Lock 50 Object (sample plate)
52 Membrane 54 Test Print Unit 56 Maintenance Unit 60 Monitor Unit 62 Dispensing Position Specification Unit 64 Dispensing Control Unit 65 Table Drive Mechanism 66 Dispensing Control Unit 68 Dispensing Position Information Creating Unit a Base Point b Reference Point

Claims (2)

A dispensing mechanism in which a dispensing element for dropping the sample or reagent is removable, an image reading device for reading the lower image, and an object to which the sample or reagent is dispensed is supported on the upper surface, and in a horizontal plane. A movable table that moves and positions the object at least at a dispensing position below the dispensing element and an image reading position below the image reading device; and at a predetermined position on the movable table by the dispensing mechanism. A calibration unit that detects a dispensing position based on an image read by the image reading device when dispensing is performed, and calibrates the dispensing position based on a base point serving as a reference on the movable table that is simultaneously read. In a dispensing device with
The dispensing mechanism has an ejection port at one end and the dispensing element that ejects a sample or a reagent;
A holding unit that is provided in the main body of the dispensing device and holds the dispensing element in a detachable manner with its discharge port facing downward;
A dispensing liquid container disposed on an upper portion of the dispensing element held by the holding unit and communicating with the dispensing element;
An air introduction head disposed at the upper part of the dispensing liquid container to communicate with the dispensing liquid container and adjust the pressure in the dispensing liquid container;
A base end portion is rotatably supported by the main body, an arm holding the air introduction head at one end portion, and the air introduction via the dispensing liquid container on the dispensing element held by the holding portion. An arm mechanism including a lock mechanism that takes a state in which the arm is fixed to the main body and a state in which the fixed state is released so that the head is fixed;
A dispensing device characterized by comprising:   The dispensing apparatus according to claim 1, wherein the dispensing mechanism includes a plurality of the dispensing elements, and the calibration unit calibrates each of the dispensing elements.

Images