JP2003247983A - Target substance mass spectrometric system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a target substance mass spectrometric system capable of performing mass spectrometry with respect to many kinds of target substances effectively and at a low cost. <P>SOLUTION: By using a measurement chip made by specifically bonding target substances to a multitude of fixed specimens fixed on a surface of a substrate, electric-field kinetic energy is given to molecular ions of the target substance ionized by irradiating each target substance with laser light thereby performing mass detection to fix the target substance. It is made possible to selectively and communicatingly connect a multitude of suction members, dispensing members, and suction/discharge members, agreeing with the number of fixed specimens fixed on the substrate with a metallic thin film formed thereon. By using the respective suction members, a prescribed amount of each fixed specimen solution is dispensed onto the substrate. After the solution of each target substance is sucked by each suction member into a corresponding suction/ discharge member, with the suction/discharge member communicating with the dispensing member, the suction/discharge member is activated to dispense the target substance solution to the fixed specimen solution fixed on the substrate for specific bonding reaction. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a target substance mass spectrometric system for ionizing and mass spectrometrically analyzing a target substance specifically bound to a fixed sample fixed on a measurement chip.

[Problems to be Solved by the Invention]

MALDI-TOF (Matrix-Assisted Laser Desorption I) is used as a mass spectrometer for identifying a target substance from its mass.
onization, Time Of Flight) method is known. This M
The ALDI-TOF method irradiates the target substance with laser light to ionize it without destroying its molecular structure, imparts constant kinetic energy to the generated target substance ions, and causes them to fly for a predetermined distance to reach the detector. Is a method of identifying the target substance by measuring the molecular mass of the target substance.

However, in the above-mentioned MALDI-TOF method, when it is applied to the use of analyzing a specific target substance from a complex mixed substance such as blood collected from a living body,
If the ratio of the target substance of interest is extremely low,
Since it is extremely difficult to efficiently ionize only the target substance of interest with laser light, mass spectrometry cannot be performed with high accuracy.

This drawback is that a specific binding substance for efficiently capturing a target substance, such as an antibody, an antigen, an enzyme, an oligonucleotide (DNA, RNA), etc., is immobilized on the measurement chip, and the specific binding substance is fixed. Although it can be solved by increasing the concentration of the target substance by specifically binding the target substance to the binding substance and then performing mass spectrometry, the number of specific binding substances that can be fixed in a conventional measurement chip is, for example, 8 samples. It was extremely small, and the number of target substances that could be analyzed in a single analysis operation was limited. Therefore, the analysis work efficiency is poor,
The analysis work took much time and the analysis cost increased.

The present invention has been invented to solve the above-mentioned conventional drawbacks, and the problem to be solved is to enable mass spectrometry of many kinds of target substances efficiently and at low cost. It is to provide a target substance mass spectrometry system.

Another object of the present invention is to provide a target substance mass spectrometry system capable of performing analysis work efficiently and highly accurately by fixing a large number of target substances at a high concentration on a measurement chip used for mass spectrometry. To provide.

[0007]

The present invention uses a measurement chip in which a target substance is specifically bound to a large number of fixed samples fixed on the surface of a substrate, and each target substance is irradiated with laser light to be ionized. Target substance mass spectrometry system that identifies the target substance by applying electric field kinetic energy to the target substance molecular ions to detect the mass, and a large number of aspirations corresponding to the number of fixed samples fixed on the substrate on which the metal thin film is formed. The member and the dispensing member and the suction and discharge member are selectively connected so that they can communicate with each other, and in a state where the suction member and the suction and discharge member are connected, each suction member collects in each reservoir of the fixed sample solution container. After each fixed sample solution is sucked by the corresponding suction / discharge member, the suction / discharge member is operated in a state where the suction / discharge member and the dispensing member are in communication with each other, and each fixed sample solution is placed on the substrate. After dispensing by volume, with each suction member connected to the suction / discharge member, suck each target substance solution stored in each reservoir of the target substance solution container by each suction member to the corresponding suction / discharge member. After that, the suction / discharge member is operated in a state where the suction / discharge member and the dispensing member are in communication with each other, and a predetermined amount of the target substance solution is dispensed to the fixed sample solution fixed on the substrate to cause a specific binding reaction. It is characterized in that a measuring chip manufacturing device for manufacturing a measuring chip is provided.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings. 1 to 8, the target substance mass spectrometry system 1 includes a measurement chip manufacturing apparatus 3 and a mass spectrometry apparatus 5.

First, the measurement chip manufacturing apparatus 3 will be described. The main body frame 7 in the measurement chip manufacturing apparatus 3 is described.
The suction / discharge device 9 is arranged on the right side of the drawing, and the movable body 11 of the suction / discharge device 9 includes an X-axis drive mechanism, a Y-axis drive mechanism, and a Z-axis drive mechanism.
It is reciprocally moved in a three-dimensional direction by an axial drive mechanism (neither is shown).

The above-mentioned drive mechanism for each axis is a feed screw drive mechanism composed of a feed screw connected to a servo motor and a nut provided on a movable body of each axis, and a pair of rotations one of which is connected to the servo motor. A belt drive mechanism or a servomotor in which a part of the belt stretched around the body is fixed to the movable body of each axis may be configured by a linear motor including a stator and a movable body provided on the movable body.

The movable body 11 has an axis line in the vertical direction, and the suction needles 13 are arranged at predetermined intervals in the X-axis direction and the Y-axis direction.
For example, 96 suction needles 13 arranged in an 8 × 12 matrix are provided. Each of the suction needles 13 has a tip end portion of each container portion 14a of the container body 14 placed on the body frame 7.
The movement is controlled so as to face. In each of the reservoirs 14a of the container body 14, the same kind or different kinds of proteins or peptides, enzymes, oligonucleotides (DNA, R, etc.) spotted on a substrate 37 of a measuring chip 35 described later are used.
The target substance solution which specifically reacts with the fixed sample solution such as NA) or the fixed sample 2 spotted on the measurement chip 35 is stored.

The base end of each suction needle 13 is connected to the suction / discharge switching member 15 via a pipe 17. The suction / discharge switching member 15 is a fixed plate (not shown) in which a suction unit and a discharge unit, the number of which is equal to the number of the suction needles 13, are provided adjacent to each other.
And a suction / ejection unit that is airtight with respect to the fixed platen and is supported so as to move at a distance according to the arrangement interval of the suction unit and the ejection unit, and that selectively communicates with each suction unit and the ejection unit. It has a switching board (not shown).

A pipe 17 connected to the suction needle 13 is connected to the suction portion of the stationary platen. Further, a pipe 23 connected to a dispensing device 21 described later is connected to the discharge part. Further, a pipe 27 connected to the suction / discharge device 25 is connected to the suction / discharge unit of the switching board.

The suction / discharge device 25 is composed of, for example, the same number of syringes 25a as the suction needles 13, and sucks the fixed sample solution and the target substance solution accumulated in each reservoir 14a into the syringe 25a as the piston reciprocates. At the same time, the aspirated fixed sample solution or target substance solution is discharged to the dispensing device 21. The amount of suction and the amount of discharge of the fixed sample solution and the target substance solution are appropriately set by the moving stroke of the piston. The stroke of the piston may be set so that the discharge amount to the dispensing device 21 is, for example, 0.5 to 10 μl, preferably 5 μl.

The fixed sample solution contains fixed sample 2 such as protein, peptide, enzyme or oligonucleotide, for example, PB.
It is a solution dissolved in S (0.14 M sodium chloride, 0.01 M phosphate buffer, adjusted to pH: 7.2).
The target substance solution is a substance that is specifically bound to the above-mentioned fixed sample 2 and analyzed, and is a solution dissolved in the same manner as above.

In the fixed sample solution and the target substance solution, the matrix is mixed with either one to accelerate the ionization described later. As the matrix to be mixed, the target substance 4 is a protein or peptide (molecular weight: 200
0 or more), sinapinic acid, peptide (molecular weight 2
000 or less), 2.5-cihydroxybenzoic acid (DHBA), 2- (4-hydroxyphenylazo) benzoic acid (HABA) for proteins, and 2-aminobenzoic acid for oligonucleotides. , 3-hydroxypicolinic acid is suitable. These matrices promote conversion into thermal energy when irradiated with laser light in the mass spectrometric operation described later.

On the left side of the main body frame 7 in the drawing, a dispensing device 2 is provided.
1 is placed. The movable body 29 of the dispensing device 21 is controlled to move in three dimensions by a drive mechanism (not shown) similar to the X-axis, Y-axis, and Z-axis drive mechanisms of the suction / discharge device 9. The lower surface of the movable body 29 has an axis line in the vertical direction, and for example, is approximately 100 to 1000 μm in the X-axis and Y-axis directions.
A large number of dispensing needles 31 arranged in an 8 × 12 matrix are attached at intervals of. Each of the dispensing needles 31 has a distal end surface having a diameter of about 500 to 2000 μm, and the pipe 23 described above is connected to the proximal end portion thereof.

The tip of each dispensing needle 31 is controlled to move selectively so as to oppose a large number of measuring chips 35 set in a measuring chip holder 33 provided in the dispensing device 21.

Each measuring chip 35 is a substrate 3 such as a slide glass or a plastic plate of polyethylene or polypropylene.
7 has a structure in which an elastic plate 39 made of silicon rubber is laminated on top. As a suitable example of the substrate 37, a slide glass is desirable, and a metal thin film 37a of gold, silver, aluminum or the like is formed on the adhered surface (surface) to form an electrode of the analysis unit 67 in the mass spectrometer 5 described later. is doing. Elastic plate 3
9 have cells 39a of the same number and array (8 × 12 matrix) as the dispensing needles 31, and at least the substrate 3
The surface relative to 7 is polished and smoothed to improve the adhesion to the substrate 37.

Regarding the measuring chip manufacturing apparatus 3, Japanese Patent Application No. 2001-34138 and Japanese Patent Application No. 2001-2773.
Since it is described in detail in No. 1, its details are omitted.

Explaining the above-mentioned measuring tip holder 33, the measuring tip holder 33 is fixedly or removably attached to the main body frame 7 on the dispensing device 21 side. The support plate 41 constituting the substrate holding member of the measuring chip holder 33 has, for example, a longitudinal direction 4 oriented in the left-right direction shown in the figure.
The four substrates 37 are of a size capable of being arranged at appropriate intervals in the longitudinal orthogonal direction (front-back direction), and four downward recesses 43 having a shape corresponding to the planar shape of the substrates 37 are formed on the upper surface thereof. It is provided at an appropriate interval in the direction. The support plate 41 engages and holds the lower portion of the substrate 37 in each downward recess 43.

Further, notches 45 are formed in the support plate 41 located in each of the downward recesses 43, and a finger or the like is inserted into each of the notches 45 to engage the substrate 37 in the downward recess 43. Allows removal of.

A base end portion of a holding plate 47 which constitutes an elastic member holding member is rotatably supported on the left end portion of the support plate 41 shown in the figure. That is, a bearing portion 49 is provided at the left and right front and rear ends of the support plate 41, and the bearing portion 49 pivotally supports a shaft support portion 51 provided at the left and right front and rear ends of the holding plate 47. 47 is rotated between a position that covers the upper surface of the support plate 41 and a position that is separated.

An upward recess 53 having a size corresponding to the downward recess 43 is formed on the bottom surface of the holding plate 47 (relative to the support plate 41) when the holding plate 47 is rotated to the upper surface side of the support plate 41. Are formed so as to face the downward recesses 43, and the elastic plate 39 forming a part of the measuring tip 35 is engaged and held in the upward recesses 53.

The holding plate 47 corresponding to the upward recess 53 is provided with a large number of openings 47a corresponding to the cells 39a of the elastic plate 39 held in the upward recess 53, respectively.

An opening / closing plate 55 is provided on the upper surface of the holding plate 47, and is approximately 1/1 of the space between the openings 47a of the holding plate 47 in the left-right direction.
Two widths are supported so as to be movable in the left-right direction. The opening / closing plate 55 is formed with a large number of slits 55a corresponding to the respective openings 47a when the holding plate 47 is moved to the left side in the drawing, and the slits 55a and the openings 47a are formed.
While each cell 39a in the elastic plate 39 is exposed to the outside via the holding plate 47, when the opening / closing plate 55 is moved to the right side in the drawing with respect to the holding plate 47, the respective slits 55a are formed into the holding plate 47 between the openings 47a. Then, the cell 39a and the opening 47a at the corresponding positions are closed.

As a structure for slidably supporting the opening / closing plate 55 with respect to the holding plate 47, support plates provided at both ends in the longitudinal direction of the holding plate 47 with the opening / closing plate 55 placed on the upper surface of the holding plate 47. In addition to the structure in which each end of the opening / closing plate 55 is movably supported by 57, each end of the opening / closing plate 55 in the front-rear direction can be bent to have a U-shaped cross section and can be moved to each end of the holding plate 47 in the front-rear direction. Or a slit 55b having a length such that the width in the left-right direction shown in the figure corresponds to the movement width of the opening / closing plate 55 is formed at each end of the opening / closing plate 55 in the front-rear direction. Any structure in which an engaging member such as a stepped shaft or a stepped screw to be inserted is provided on the holding plate 47 and the opening / closing plate 55 is movably supported with respect to the holding plate 47 may be used.

Actuating arms 59 are formed at the front and rear ends on the right side of the opening / closing plate 55 so as to project outward, and engaging holes 59a are provided in each actuating arm 59. The engaging hole 59a of each actuating arm 59 can be engaged with an engaging portion 61a provided in an actuating member 61 such as an electromagnetic solenoid or an air syringe attached to the front and rear ends of the supporting plate 41 shown in the figure. The opening / closing plate 55 is opened / closed with respect to the holding plate 47 by the operation of the operating member 61.

The locking member 6 is provided on the right side of the support plate 41 in the figure.
3 is rotatably supported. The locking member 63 is the support plate 4
1. A locking arm portion 63a that abuts the illustrated right end portion of the holding plate 47 that is rotated so as to cover the upper surface of 1 over the entire front-rear direction, and hangs down at both end portions in the front-rear direction of the locking arm portion 63a. Support arm portion 63 that is pivotally supported by the support plate 41
b. The shaft support arm portion 63b has elastic plates 39 held in the upward recesses 53 of the holding plate 47 when the locking arm portion 63a abuts against the upper surface of the right end portion of the holding plate 47 shown in the figure and locks. Is set to a length in which each substrate 37 held in each downward recess 43 of the support plate 41 is brought into close contact with.

It should be noted that when the locking member 63 is locked to the holding plate 47, the elastic plate 39 needs to be surely brought into close contact with the substrate 37, but the length of the shaft support arm portion 63b is shortened. When the locking member 63 is firmly attached to the holding plate 47,
The operability when locking and unlocking becomes poor. In order to avoid this, a biasing member (not shown) such as a leaf spring or a spring-loaded pin is provided on the relative surface of the holding plate 47 in the locking arm portion 63a.
The holding plate 47 can be urged in the closing direction by the elastic force of the urging member, and the elastic plate 39 can be brought into close contact with the substrate 37.

As shown in FIG. 10, the mass spectrometer 5 is a conventionally known MALDI-TOF type mass spectrometer, which ionizes the target substance 4 and ionizes the target substance molecule at a predetermined distance.
The target substance 4 is analyzed by measuring the mass based on the flight time difference detected by the flight, and is composed of an ion source 65, an analysis unit 67, and a detection unit 69.

The ion source 65 is for irradiating the target substance 4 of each cell in the measuring chip 35 set on the support 71 with laser light to heat and vaporize it to generate + ions and −ions. The wavelength of light is 337
N ₂ which outputs a pulsed laser beam of nm or 355 nm
Laser, Na-YAG laser, CWCO ₂ laser, TE
A-CO ₂ laser or the like can be applied.

In the analysis unit 67, the negative electrode plate 73 is arranged in front of the support 71, and among the ions generated by applying an electric field of a predetermined voltage between the metal thin film in the measuring chip 35 and the negative electrode plate 73, The + ions are electrically attracted to the negative electrode plate 73 to fly toward the detection unit 69.

The detector 69 measures the + ion velocity based on the difference in ion arrival time due to the difference in the mass-to-charge ratio m / z value of the flying ions, detects the flight velocity of the + ions by the Doppler effect, and analyzes the mass. .

The principle of mass spectrometry using the target substance ions is as follows. That is, a constant acceleration voltage V for ions
When the ion mass is m, the ion velocity is ≡, the charge number of the ion is z, and the electric mass is e, the kinetic energy imparted to the ion is v is obtained by the equation (1).

Now, if the flight distance L of ions due to kinetic energy is constant, the flight time t at velocity v is L / v,
The flight time will differ depending on the m / z of the ion, and the mass of the target substance ion can be obtained by measuring the flight time.

[0037]

[Equation 1]

Next, a sample analysis method by this system will be described. First, the method for producing the measuring chip 35 will be described. Prior to producing the measuring chip 35, the movable body 11 is moved in a state in which each suction needle 13 and each syringe 25a of the suction / discharge device 25 are connected by the suction / discharge switching member 15. After the movement is controlled and a large number of suction needles 13 are retracted into the respective reservoirs 14a of the container body 14 in which a fixed sample solution such as an antibody having specificity for the target substance 4 is stored, the pistons are moved in the suction direction. The solution moves and the fixed sample solution in the reservoir is sucked and stored in the syringe 25a.

After the above-mentioned suction action, the suction / discharge switching member 1
5 of the syringe 2 of the suction and discharge device 25 by moving the switching board 5
5a and each dispensing needle 31 are connected.

On the other hand, in the state where the holding plate 47 is rotated to the open side with respect to the support plate 41, each downward recess 4 of the support plate 41.
3 in which the substrate 37 is provided, and the upward recesses 53 of the holding plate 47.
After setting the elastic plate 39 therein, the holding plate 47 is rotated with respect to the support plate 41 in the closing direction to lock the locking member 63 at the tip of the holding plate 47.

At this time, the holding member 47 is biased toward the support plate 41 side by the locking of the locking member 63, and the elastic plate 39 is elastically deformed to be brought into close contact with the substrate 37. Further, when the holding plate 47 is rotated toward the closing side with respect to the support plate 41, the engaging portion 61a of the operating member 61 is engaged with the engaging hole 59a.
Further, the cells 39a are closed by positioning the opening / closing plate 55 between the slits 55a moved to the right on the upper surface of the holding plate 47 in the opening 47a.

Furthermore, since the relative surface of the elastic plate 39 to the substrate 37 has been polished and smoothed in advance, the substrate 3
The elastic plate 39 is brought into close contact with 7 at a high airtightness to prevent the solution dispensed in each cell 39 from leaking and preventing mutual contamination.

In the above state, the actuating member 61 is actuated to cause the opening / closing plate 55 to align the slits 55a with the openings 47a of the holding plate 47, thereby exposing the cells 39a of the elastic plate 39 to the outside. .

Next, in the above state, the movable body 29 is controlled to move to expose the respective dispensing needles 31, and the slits 55a and the slits 55a and the slits 55a are provided for the cells 39a in the elastic plate 39 provided in the first row in the front-rear direction. After they are made to face each other through the opening 47a, the movable body 29 is lowered to move the tip end portion of each dispensing needle 31 into each cell 39a. In this state, the piston in each syringe 25a is slightly moved to discharge the fixed sample solution stored in the syringe 25a to the side of each dispensing needle 31 and dispense into each cell 39a.

At this time, the moving amount of the piston in the syringe 25a is controlled so that the fixed sample solution stored in the cell 39a is 0.5 to 10 μl, preferably 5 μl. Further, as described above, since the elastic plate 39 is in close contact with the upper surface of the substrate 37 with high airtightness, the fixed sample solution stored in the cell 39a leaks and is stored in each cell 39a. To prevent cross-contamination of the fixed sample solution of.

Next, the movable body 29 is moved upward to extract the respective dispensing needles 31 from the cells 39a of the elastic plate 39 in the first row in the front-rear direction, and then the movable body 29 is moved in the front-rear direction. Each cell 39 in the elastic plate 39 in the second row in the direction
After facing the a, the movable body 29 is moved downward and the dispensing needles 31 are moved into the cells 39a to move the pistons of the syringes 25a in the second row in the front-rear direction as described above. A fixed amount of the fixed sample solution is dispensed into each cell 39a of the elastic plate 39 in the eye.

By repeating the above-mentioned operation, each elastic plate 39 that comes into close contact with each substrate 37 set on the support plate 41.
A fixed amount of the fixed sample solution is dispensed into the cell 39a in FIG. 4 to produce four measurement chips 35, and then the operating member 61 is moved back to move the opening / closing plate 55 to the right in the figure to move each slit 55a. Each cell 39a is closed by locating the opening / closing plate 55 between them in each opening 47a.

This prevents the fixed sample solution stored in each cell 39a of the elastic plate 39 of the measuring chip 35 from being denatured or deactivated by drying when it is a protein, and the liquid phase described later is used. The measuring chip 35 capable of reliably performing the specific binding with the target substance 4 therein can be manufactured.

Next, a method for specifically binding the target substance 4 to the fixed sample 2 will be described. Prior to dispensing the target substance solution to the fixed sample 2 in the measuring chip 35, the measuring chip 3
The many suction needles 13, the suction / discharge switching member 15, the suction / discharge device 25, the dispensing needle 31, and the pipes 17, 23, and 27 that connect these, which were used when producing 5, are washed.

As a method for cleaning the fixed sample 2 attached to these, first, suction is performed with a recovery container (not shown) placed on the main body frame 7 corresponding to the suction / discharge device 9 and the dispensing device 21, respectively. The suction switching device 15 is operated while switching the respective flow paths by the discharge switching member 15 to operate the suction needle 13, the suction / discharge switching member 15, the suction / discharge device 25 and the dispensing needle 31, and the pipes 17, 23, 27 connecting these.
Excess fixed sample solution in each suction needle 13 and each dispensing needle 3
From 1 to each of the collection containers are discharged and collected.

Next, the main body frame 7 on the suction / ejection device 9 side
Each dispensing needle 3 is placed in the cleaning solution dissolver (not shown) placed on the top.
1 is immersed, the suction / discharge device 25 is operated to suck the cleaning liquid into the syringe 25a, and then the suction / discharge switching member 15 switches the flow path to the suction needle 13 side and the dispensing needle 31 side in this order. The operation of discharging the cleaning liquid accumulated by operating the discharging device 25 from the suction needles 13 or the dispensing needles 31 is repeated a plurality of times to suck the suction needles 13,
Suction and discharge switching member 15, suction and discharge device 25, dispensing needle 31
And, the fixed sample solution attached to the pipes 17, 23, and 27 connecting them is washed.

As the cleaning liquid used for these cleaning,
After washing with 0.005 to 0.1% Tween20 aqueous solution, ultrapure water, and PBS in this order, the piston of each syringe 25a of the suction / discharge device 25 is moved to move the internal air to each suction needle 13 and each Discharge from the dispensing needle 31 and the suction needle 1
3. The suction / discharge switching member 15, the dispensing needle 31, and the pipes 17, 23, and 27 connecting them are dried.

When the next measuring chip 35 is produced, the target substance attached to the suction needle 13, the suction / discharge switching member 15, the dispensing needle 31 and the pipes 17, 23 and 27 connecting them are fixed as described above. It may be washed in the same manner as the sample washing method.

After the cleaning process described above, when the container body 14 accumulating the target substance 4 to be analyzed in each reservoir is set on the main body frame 7 corresponding to the suction / discharge device 9, the measuring chip 35 is produced. Similarly to the above, the movable body 11 is controlled to move and the suction needles 13 are moved to the container body 1 in which the target substance 4 is stored.
4, and then the suction / discharge device 2
Each piston in 5 is suctioned to suck the target substance solution in the reservoir into the syringe 25a and store it.

After the suction operation of the target substance solution described above, the suction / discharge device 25 is moved by moving the switching plate of the suction / discharge switching member 15.
After switching the flow paths so that each syringe 25a and each dispensing needle 31 are connected, the movable body 29 is controlled to move and each dispensing needle 31 is held by the measuring chip holder 33, for example, in the front-back direction. The cells 39a of the elastic plate 39 in the measurement chip 35 of the first row are made to face each other.

At this time, the actuating member 61 of the measuring tip holder 33 is actuated to move the opening / closing plate 55 to expose the cell 39a of the elastic plate 39 of each measuring tip 35 produced to the outside.

Next, in the above state, the movable body 29 is moved downward so that the respective dispensing needles 31 enter the respective cells 39a, and then the respective pistons of the suction / discharge device 25 are moved in the discharge direction by a predetermined amount. Then, a predetermined amount of the target substance solution stored in the syringe 25a is discharged from each dispensing needle 31 into each cell 39a.

After a predetermined amount of the target substance solution is discharged into the cell 39a of the elastic plate 39 of each measuring chip 35 set in the measuring chip holder 33 by repeating the above operation, the actuating member 61 is moved back to open and close. By moving the plate 55 in the closing direction, each cell 3 of the elastic plate 39 in each measuring chip 35 is moved.
9a is closed and left in this state for about 30 minutes to 2 hours to specifically bind the fixed sample 2 stored in each cell 39a of the elastic plate 39 in each measurement chip 35 and the target substance 4.

During this reaction, since each cell 39a of the elastic plate 39 in each measurement chip 35 is shielded from the outside air by the opening / closing plate 55, the fixed sample solution and the target substance solution stored in each cell 39a are dried. Can be prevented and specific binding of both can be ensured.

Next, as shown in FIG. 9, the measuring chip 35 in which the target substance 4 is specifically bound to the fixed sample 2 is dried, and then elastic from the substrate 37 of the measuring chip 35 taken out from the measuring chip holder 33. The plate 39 is removed, and then the non-fixed area including the empty cell in the measurement chip 35 is blocked.

As a method for drying the measuring chip 35 when a polynucleotide such as DNA is used as a fixed sample,
A UV cross-linker (UVC500 manufactured by Hoefer) is used to irradiate the surface of the measurement chip 35 with ultraviolet light of about 50 to 120 mj to dry and fix it.

As the blocking processing, 1 to
1.5 wt% succinic anhydride, 90% by volume N-methylpyrocinone, 10% by volume 0.2 M sodium borate solution (p
(H: 8.0) The pH is adjusted to 5 to 6 by immersing the entire measuring chip 35 on which the polynucleotide is immobilized in the block solution for about 15 seconds.

Then, the measuring chip 35 taken out from the block solution is immersed in hot water at 95 to 99 ° C. and left for about 60 seconds. Then, the measuring chip 35 is immersed in ethanol, and then the surface of the measuring chip 35 is air-dried.

As a blocking treatment method when a protein is used as a fixed sample, an ethanolamine solution (pH: 8 to 9) or a solution prepared by adjusting about 0.1 to 3% of casein or bovine serum albumin is used. , As well
Dip for about 1 hour to block. That is, the measurement chip 35 was placed in a block solution adjusted to a final concentration of 50 mmol / l ethanolamine aqueous solution (pH: 9.0).
It is immersed for about a time and then washed with water to react with a target substance for specific binding.

As a result, the presence ratio of the target substance 4 can be increased by specifically binding the target substance 4 to the fixed sample 2, and the ionization treatment described later can be efficiently performed.

Next, as shown in FIG. 10, the measuring chip 35 with or without the elastic plate 39 attached is removed from the support base 7.
After setting on 1, the substrate 37 in the measuring chip 35
The target substance 4 fixed to each cell 39a is irradiated with laser light in a state where an electric field is generated between the metal thin film and the negative electrode plate 73, which is grounded, and is heated and vaporized. Generate the + ion and-ion of the target substance molecule.

Among the ionized target substances 4, + ionized target substance ions are given kinetic energy due to the electric field between the metal thin film and the negative electrode plate 73, so that the negative electrode plate 7 is exposed.
It flies to the 3 side and is detected by the detection unit 69.

Then, the calculation unit 73 calculates the ion velocity based on the arrival time difference of the ions in the detection unit 69,
The target substance 4 is identified by measuring the mass of the target substance molecule based on the above-mentioned formula 1.

When the target substance 4 is mixed with the matrix, the heating of the target substance 4 by the irradiation laser beam is promoted, so that the target substance 4 can be vaporized and ionized in an extremely short time.

In the present embodiment, the target substance 4 to be subjected to mass spectrometry is reacted with the fixed sample fixed to the measurement chip to specifically bind to the target substance 4 to increase the abundance ratio of the target substance 4, thereby efficiently targeting the target substance 4. Can be ionized, and mass spectrometry can be efficiently performed.

At least 96 fixed samples 2 can be fixed on the measurement chip 35 to specifically bind the target substance 4, and the mass spectrometric operation can be performed efficiently and the measurement operation can be performed at low cost.

[0072]

INDUSTRIAL APPLICABILITY The present invention enables efficient mass spectrometric analysis of many kinds of target substances. In addition, a large number of target substances can be fixed at a high concentration on the measurement chip used for mass spectrometry, and the analysis work can be performed efficiently and highly accurately.

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing an example of an analysis process of a target substance mass spectrometry system.

FIG. 2 is an explanatory diagram showing a measuring chip manufacturing apparatus.

FIG. 3 is an explanatory diagram showing a suction side in the measuring chip manufacturing apparatus.

FIG. 4 is an explanatory diagram showing a dispensing side in a measurement chip manufacturing apparatus.

FIG. 5 is an explanatory view showing a tip holder.

FIG. 6 is an explanatory view showing a state in which an opening / closing plate of the chip holder is opened.

7 is a cross-sectional view taken along the line AA of FIG.

8 is a cross-sectional view taken along the line BB of FIG.

FIG. 9 is an explanatory diagram showing a fixed state of a fixed sample and a target substance on a substrate.

FIG. 10 is an explanatory diagram showing the analysis principle of the mass spectrometer.

[Explanation of symbols]

2-Fixed sample, 3-Measurement chip preparation device, 4-Target substance, 5-Mass spectrometer, 9-Suction and discharge device, 21-Dispensing device, 25-Suction and discharge member, 35-Measurement chip, 37-
substrate

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Emiko Moriyama             20-9 Sanbonmatsucho, Atsuta-ku, Nagoya             Within The Electronics Co., Ltd. (72) Inventor Yasuhiro Fukao             20-9 Sanbonmatsucho, Atsuta-ku, Nagoya             Within The Electronics Co., Ltd. (72) Inventor Hidekatsu Yoneda             20-9 Sanbonmatsucho, Atsuta-ku, Nagoya             Within The Electronics Co., Ltd. F term (reference) 2G052 AA28 AD06 AD26 AD46 CA04                       CA13 CA30 CA31 DA05 FC05                       FD06 GA24 HC04 HC07 HC08                       HC10 JA06 JA09

Claims (8)

[Claims] 1. A measurement chip in which a target substance is specifically bound to a large number of fixed samples fixed on the surface of a substrate is used, and each target substance is irradiated with a laser beam to ionize the target substance molecular ion to have an electric field kinetic energy. In a target substance mass spectrometry system that identifies a target substance by giving a mass detection, a large number of suction members and dispensing members that match the number of fixed samples fixed on a substrate on which a metal thin film is formed, and suction and discharge A fixed sample solution stored in each pool of the fixed sample solution container by each suction member in a state where the suction member and the suction / discharge member are connected. After the suction / discharge member has been sucked, the suction / discharge member and the dispensing member are in communication with each other to operate the suction / discharge member to dispense a fixed amount of each fixed sample solution onto the substrate, and then the suction unit In a state where the suction and discharge member connected to,
The target substance solution stored in each reservoir of the target substance solution container by each suction member is sucked by the corresponding suction and discharge member, and then the suction and discharge member and the dispensing member are in communication with each other. The target substance mass spectrometric system provided with a measurement chip production device for producing a measurement chip by dispensing a predetermined amount of a target substance solution into a fixed sample solution fixed on a substrate to cause a specific binding reaction. 2. A suction / dispensing solution by washing excess liquid adhering to the suction member, the dispensing member and the suction / discharging member by a cleaning means when switching the suction / discharging of the fixed sample solution and the target substance solution by the measuring chip manufacturing apparatus. The target substance mass spectrometric system according to claim 1, wherein the switch is made possible. 3. The mass spectrometry system for a target substance according to claim 1, wherein the measuring chip comprises a substrate on which a metal thin film is formed, and an elastic body that is in close contact with the substrate surface and has a large number of cells provided on a matrix. 4. A measuring chip holder for holding a measuring chip when a fixed sample and a target substance sample are dispensed onto a substrate, a substrate holding member for holding at least one or more substrates, and the substrate holding member. An elastic body holding member that is rotatably supported with respect to the substrate holding member, has an elastic body holding portion on a surface relative to the substrate holding member, and has a large number of openings corresponding to each cell of the elastic body; The opening / closing member movably supported with respect to each other to open and close each opening, and the locking member that locks the elastic body holding member to bring the elastic body into close contact with the substrate held by the substrate holding member. Target substance mass spectrometry system. 5. The target substance mass spectrometry system according to claim 1, wherein the substrate is a slide glass having a metal thin film formed on the surface thereof. 6. The target substance mass spectrometry system according to claim 5, wherein the metal thin film is made of any one of gold, silver and aluminum. 7. The target substance mass spectrometry system according to claim 1, wherein the fixed sample and the target substance are dissolved in a buffer solution. 8. The target substance mass spectrometric system according to claim 1, wherein either one of the fixed sample and the target substance is mixed with a matrix.