WO2021234806A1

WO2021234806A1 - Sample pretreatment method, analysis method, sample pretreatment device, and analysis system

Info

Publication number: WO2021234806A1
Application number: PCT/JP2020/019732
Authority: WO
Inventors: 友祐中谷; 幸夫老川; 重吉堀池
Original assignee: 株式会社島津製作所
Priority date: 2020-05-19
Filing date: 2020-05-19
Publication date: 2021-11-25

Abstract

A sample pretreatment method according to one aspect comprises: a step for generating an acoustic standing wave; a step for introducing a liquid sample into the acoustic standing wave; and a step for concentrating the liquid sample by holding the liquid sample at the position of the node of the acoustic standing wave. A sample analysis method according to one aspect comprises: a step for holding a liquid sample at the tip of an elongated holding member and concentrating the liquid sample; a step for placing the concentrated liquid sample on a sample plate; and a step for introducing the liquid sample into an analysis device.

Description

Sample pretreatment method, analysis method, sample pretreatment device and analysis system

The present invention relates to a sample pretreatment method, an analysis method, a sample pretreatment device, and an analysis system.

US Patent Application Publication No. 2007/0075241 (Patent Document 1) discloses a sample plate used for mass spectrometry of a sample using the MALDI method (matrix-assisted laser desorption / ionization method). The sample plate described in Patent Document 1 has a substrate and a hydrophobic coating layer formed on the surface of the substrate. The coating layer is arranged so as to cover the sample area on the surface of the substrate on which the sample is placed. The coating layer is formed with an opening for exposing the surface of the substrate located in the central portion of the sample region.

In Patent Document 1, a mixed solution of the sample solution and the matrix is dropped into the opening of the sample plate. Since the circumference of the opening is surrounded by a coating layer having hydrophobicity, the droplets of the mixed solution are fixed to the opening. By drying the droplets and evaporating the solvent, the sample solution is concentrated and a mixed crystal of the sample and the matrix can be obtained.

U.S. Patent Application Publication No. 2007/0075241

By obtaining high-concentration crystals in the sample pretreatment, it is possible to perform high-sensitivity analysis even for a small amount of sample. However, the above-mentioned sample pretreatment method using a sample plate has a problem that it is difficult to increase the concentration ratio of the sample solution. This is because the sample solution in contact with the surface of the substrate at the opening of the sample plate remains without evaporating. Further, there is a problem that droplets cannot be formed on the sample plate depending on the type of sample. This is because when the oil content of the sample is large, the sample solution cannot be applied to the hydrophobic coating layer, which makes it difficult to form droplets.

The present invention has been made to solve such a problem, and an object of the present invention is a sample pretreatment method and pretreatment that enable high-sensitivity analysis of a sample regardless of the type of the sample. It is to provide a processing device.

The sample pretreatment method according to the first aspect of the present invention includes a step of generating an acoustic standing wave, a step of introducing a liquid sample into the acoustic standing wave, and a liquid sample at the position of a node of the acoustic standing wave. It is provided with a step of concentrating the liquid sample by holding the above.

The sample pretreatment apparatus according to the second aspect of the present invention includes a means for generating an acoustic standing wave, a means for introducing a liquid sample into the acoustic standing wave, and a liquid sample at the position of a node of the acoustic standing wave. It is provided with a means for concentrating a liquid sample by holding the above.

According to the present invention, it is possible to provide a sample pretreatment method and a pretreatment apparatus capable of analyzing a sample with high sensitivity regardless of the type of the sample.

It is a block diagram which shows the whole structure of the analysis system which concerns on Embodiment 1. FIG. It is a flowchart for demonstrating the analysis method which concerns on Embodiment 1. It is a flowchart for demonstrating the process procedure of a pre-processing process. It is a figure which shows the structural example of the pretreatment apparatus schematically. It is a figure for demonstrating the procedure of the process of concentrating a sample solution, and the process of placing a sample solution on a sample plate. It is a schematic diagram which shows the structural example of the analyzer. It is a flowchart for demonstrating the processing procedure of an analysis process. It is a figure for demonstrating the processing procedure of the general sample pretreatment method. It is a figure for demonstrating the processing procedure of the pretreatment process which concerns on Embodiment 2. FIG. It is a figure which shows an example of the mass spectrum created in the analysis process. It is a figure which shows the other configuration example of the pretreatment apparatus schematically.

Hereinafter, embodiments of the present disclosure will be described in detail with reference to the drawings. In the following, the same or corresponding parts in the figure are designated by the same reference numerals, and the description thereof will not be repeated in principle.

[Embodiment 1]
<Analysis system configuration>
FIG. 1 is a block diagram showing an overall configuration of the analysis system according to the first embodiment. The analysis system 100 according to the first embodiment includes a pretreatment device 10 and an analysis device 20.

The pretreatment device 10 is a device for pretreating a compound as a sample. The pretreatment of the sample includes a treatment of purifying (mainly desalting) the sample and a treatment of concentrating the sample solution obtained by dissolving the sample in a solvent. The pretreatment device 10 can be used for the treatment of concentrating the sample solution. The sample may be a solid phase or a liquid phase. The sample solution corresponds to one embodiment of the "liquid sample".

The analyzer 20 is an apparatus for analyzing the pretreated sample. The analyzer 20 is, for example, a mass analyzer for measuring the mass (mass: m) of an atom or molecule contained in a sample, or a spectrum for measuring a spectrum emitted or absorbed by an atom or molecule contained in a sample. It is a spectroscopic analyzer. These analyzers can be suitably used for identification and qualitative use of biopolymers such as proteins.

The mass spectrometer includes a liquid chromatograph (High Performance Liquid Chromatography: HPLC), a liquid chromatography-Mass Spectrometry (LC-MS), a gas chromatograph (Gas Chromatography: GC), and a gas chromatograph mass spectrometer. (Gas Chromatography-Mass Chromatography: GC-MS), Matrix Assisted Laser Deposition / Ionization Time-of-Flight Mass Spectrometer: MALDI-TOF-MS, and ICP mass An analyzer (Inductively Coupled Plasma Mass Spectrometer) is included.

The spectrophotometer includes a Fourier transform infrared spectrophotometer (FT-IR; Fourier Transform Infrared Spectroscopy), a Raman spectrophotometer, and an ultraviolet-visible spectrophotometer.

The analyzer 20 includes a sample introduction unit 22 for introducing a pretreated sample, a measurement unit 24 for measuring the mass or spectrum of an atom or molecule contained in the sample, and a measurement acquired by the measurement unit 24. It has a processing unit 26 for processing data.

In the first embodiment, the analyzer 20 is a mass spectrometer. More specifically, the analyzer 20 is a MALDI-TOF-MS. The MALDI method (matrix-assisted laser desorption / ionization method) is a method in which the surface of a condensed phase such as a crystal or liquid as a sample is irradiated with pulsed laser light to separate the sample molecule from the surface, and at the same time, the sample molecule is ionized. .. TOF-MS (Time-of-Flight Mass Spectrometry) is a method of accelerating ionized sample molecules by an electric field, flying them over a certain distance, and measuring the flight time to determine the molecular weight. When MALDI-TOF-MS is used for the analyzer 20, the pretreatment device 10 is configured to crystallize a mixed solution of the sample solution and the matrix as a pretreatment of the sample.

<Analysis method>
FIG. 2 is a flowchart for explaining the analysis method according to the first embodiment. As shown in FIG. 2, the analysis method according to the first embodiment includes a pretreatment step S10 and an analysis step S20. Hereinafter, the processing procedure of each step will be described in detail.

(1) Pretreatment step (S10)
FIG. 3 is a flowchart for explaining the processing procedure of the preprocessing step (S10).

With reference to FIG. 3, the pretreatment steps include a step of preparing the sample solution (S11), a step of concentrating the sample solution (S12), a step of placing the sample solution on the sample plate (S13), and a sample solution and a matrix. A step of mixing with (S14), a step of crystallizing the mixed solution (S15), and a step of introducing the sample into the analyzer 20 (S16) are provided.

In the step of preparing the sample solution (S11), the sample is dissolved in a solvent to generate a sample solution. The sample is, for example, a polymer, protein or peptide. The sample is pre-purified to remove non-volatile components. The solvent is an aqueous or organic solvent. As the solvent, for example, water, acetonitrile / water, chloroform, tetrahydrofuran and the like can be used.

In the step of concentrating the sample solution (S12), the sample solution is concentrated by evaporating the solvent of the sample solution. This step is carried out using the pretreatment apparatus 10 (see FIG. 1). FIG. 4 is a diagram schematically showing a configuration example of the pretreatment device 10.

With reference to FIG. 4, the pretreatment device 10 has an ultrasonic levitation device 12 and two

cameras

13 and 14. The pretreatment device 10 is configured to hold the droplets in the space in a non-contact manner by suspending the droplets of the sample solution using ultrasonic waves. In FIG. 4, the vertical direction of the paper surface is the Z direction, and the two directions perpendicular to the Z direction are the X direction and the Y direction.

The ultrasonic floating device 12 has an ultrasonic vibrator 120, a horn 122, a reflector 124, and an oscillator 126. The oscillator 126 receives power from the commercial power supply 128 to drive the ultrasonic oscillator 120. The oscillator 126 converts the power of the commercial power supply 128 into ultrasonic power.

The ultrasonic vibrator 120 generates ultrasonic vibration. The horn 122 is connected to the ultrasonic vibrator 120, and emits ultrasonic waves from the ultrasonic vibrator 120 toward the air. The amplitude of the ultrasonic wave is amplified by the horn 122 and transmitted.

The reflector 124 is arranged so as to face the horn 122 along the Z direction (the direction perpendicular to the paper surface). The reflector 124 reflects the ultrasonic waves transmitted from the horn 122. An acoustic standing wave W is formed between the horn 122 and the reflector 124 by ultrasonic waves and reflected waves. When a droplet is injected into the acoustic standing wave W, the droplet is held at the position P1 of the sound pressure node in the potential field of the pressure of the acoustic standing wave W formed by the sound field.

The wavelength of the acoustic standing wave W can be adjusted by the oscillation frequency of the ultrasonic vibrator 120. The wavelength of the acoustic standing wave W determines the number and position of the nodes of the acoustic standing wave W.

The two

cameras

13 and 14 are provided to indicate the position P1 of the node of the acoustic standing wave W. Specifically, the first camera 13 is arranged so as to take an image of the ultrasonic levitation device 12 from the Y direction. In the image captured by the first camera 13, the position P1 of the node of the acoustic standing wave W in the XZ plane is indicated by the coordinates (X, Z). The second camera 14 is arranged so as to take an image of the ultrasonic floating device 12 from the X direction. In the image captured by the second camera 14, the position P1 of the node of the acoustic standing wave W in the YZ plane is indicated by the coordinates (Y, Z). These two coordinates can be obtained by calculation using the oscillation frequency of ultrasonic vibration. The position P1 of the node in the three-dimensional space can be specified based on the images captured by the first camera 13 and the second camera 14.

FIG. 5 is a diagram for explaining the procedure of the step of concentrating the sample solution (S12) and the step of placing the sample solution on the sample plate (S13). In the step (S12), first, as shown in FIG. 5 (A), the acoustic standing wave W is formed by activating the ultrasonic floating device 12 to generate ultrasonic vibration. A droplet Dp of the sample solution is injected into the position P1 of the node of the acoustic standing wave W.

Specifically, since the position P1 of the node of the acoustic standing wave W is indicated on the images captured by the

cameras

13 and 14, the user (for example, the measurer) can refer to the captured images and refer to the micropipette 30. Is used to inject a droplet Dp of the sample solution into position P1. The droplet Dp has a liquid volume of about 10 μL. The maximum amount of droplet Dp that can be injected into the node position P1 depends on the specific gravity and surface tension of the solvent of the sample solution.

It should be noted that the injection of the droplet Dp can be automatically performed by using a mechanism having a robot arm to which the micropipette 30 is attached, instead of the manual operation by the user.

Next, as shown in FIG. 5B, by continuously forming the acoustic standing wave W, the droplet Dp can be stably held in a suspended state in the air. The droplet Dp is concentrated by gradually evaporating the solvent from the suspended droplet Dp.

Next, as shown in FIG. 5C, the concentrated droplet Dp is taken out from the ultrasonic levitation device 12. When the user visually recognizes the droplet Dp and determines that the concentration ratio has reached a desired value, the user takes out the droplet Dp from the position P1 of the node of the acoustic standing wave W using the micropipette 30. The process proceeds to step (S13), and the removed droplet Dp is placed on the sample plate 40 (see FIG. 5 (D)). The droplet Dp can be taken out automatically by using the above mechanism instead of the manual operation by the user.

Note that the means for specifying the position P1 of the node of the acoustic standing wave W in the three-dimensional space is not limited to the means using the images captured by the two

cameras

13 and 14. For example, in a configuration in which the injection / extraction of the droplet Dp to the acoustic standing wave W is automatically performed using a mechanism, the mechanism is taught in advance about the position P1 of the node of the acoustic standing wave W. Therefore, the droplet Dp can be injected and taken out without using the

cameras

13 and 14.

Returning to FIG. 3, in the step (S14) of mixing the sample solution and the matrix, the matrix is mixed with the droplet Dp of the sample solution placed on the sample plate. The matrix has the property of absorbing the laser beam used in the MALDI method. Typical matrices used for protein and peptide ionization are sinapinic acid (SA), 2,5-dihydroxynbenzoic acid (2,5-DHB), and α-cyano-4-hydroxyn cinnamic acid (CHCA). ) And so on.

In the step (S14), the mixed solution of the sample solution and the matrix placed on the sample plate 40 is dried to volatilize the solvent in the mixed solution and crystallize it.

(2) Analysis step (S20)
In the analysis step (S20), mass spectrometry of the pretreated sample is performed using the analyzer 20 (see FIG. 1). FIG. 6 is a schematic diagram showing a configuration example of the analyzer 20.

With reference to FIG. 6, the analyzer 20, MALDI-TOF-MS, has a sample introduction unit 22, an ionization unit 240 (ion source), a mass separation unit 250, a detector 260, and a processing unit 26. Of these, the ionization unit 240, the mass separation unit 250, and the detector 260 constitute the measurement unit 24 (see FIG. 1).

FIG. 7 is a flowchart for explaining the processing procedure of the analysis step (S20). As shown in FIG. 7, in the analysis step (S20), the step of introducing the sample plate 40 into the ionization unit 240 of the analyzer 20 (S21), the step of ionizing the sample (S22), and the mass separation of the ionized sample are performed. A step (S23), a step of detecting (S24), and a step of creating a mass spectrum (S25) are provided.

Next, the processing procedure of the analysis step (S20) will be described with reference to FIGS. 6 and 7.
In the step (S21), the sample plate 40 on which the crystal as the sample S is placed is introduced from the sample introduction unit 22 into the ionization unit 240. The sample S is in a state of being uniformly mixed with the matrix.

In the step (S22), the ionization unit 240 (ion source) ionizes the sample molecule. Specifically, the ionization unit 240 has a laser light source 242. The laser light emitted from the laser light source 242 irradiates the surface of the sample S on the sample plate 40. As the laser light, nitrogen laser light, which is ultraviolet light, is generally used.

When the surface of the sample S is irradiated with the laser beam, the matrix absorbs the laser beam and becomes an excited state. At this time, the temperature at the spot of the laser beam rises, and an evaporation phenomenon occurs. The sample S evaporates due to the thermal energy obtained from the matrix in a highly excited state. The sample molecules desorbed from the surface of the sample S are ionized by the transfer of protons while floating in the gas phase on the gas of the matrix desorbed and vaporized in large quantities. The sample molecule ionized by the ionization unit 240 is introduced into the mass separation unit 250.

In the step (S23), the mass separation unit 250 separates individual sample molecules according to their mass. In the example of FIG. 6, the mass separation unit 250 has a flight tube 252, and separates ions due to the difference in flight time when the introduced sample molecule flies inside the flight tube 252. Although the linear type flight tube 252 is shown in FIG. 6, it may be a reflector type or a multi-turn type. Further, the method of mass spectrometry is not particularly limited as long as the ions can be separated and detected.

In the step (S24), the detector 260 has an ion detector such as a multi-channel plate. The detector 260 detects the ions separated by the mass separation unit 250, and outputs a detection signal having an intensity corresponding to the number of ions incident on the detector 260. The detection signal output from the detector 260 is input to the processing unit 26.

The processing unit 26 is configured to include a processor such as a CPU and a memory, and controls the operation of the analyzer 20. The processing unit 26 executes various processes by executing the program stored in the memory. The processing unit 26 also controls the operation of the measurement unit 24 based on the data regarding the measurement conditions input from the input unit (not shown).

The processing unit 26 further analyzes the measurement data based on the detection signal output from the detector 260. In the step (S25), the processing unit 26 appropriately uses a calibration curve or the like obtained by measuring the standard substance to set the flight time corresponding to each detected ion to the mass-to-charge number ratio. : M / z) to create a mass spectrum. The processing unit 26 calculates the amount of ions detected corresponding to each peak from each peak in the mass spectrum. The amount of ion detected is quantified by the peak area, which is the area corresponding to the peak, or the peak intensity, which is the maximum value of the peak. The peak area and peak intensity can be calculated by removing noise by an appropriately known method or by smoothing the detected intensity value.

<Action effect>
Next, the action and effect of the pretreatment method according to the first embodiment will be described.

First, as a comparative example, a general sample pretreatment method in the MALDI method will be described. FIG. 8 is a diagram for explaining a processing procedure of a general sample pretreatment method.

In a general sample pretreatment method, a sample and a matrix are mixed using a solvent to prepare a sample solution. As shown in FIG. 8A, the droplet Dp of this sample solution is placed on the sample plate 40. The amount of the sample solution placed on the sample plate 40 is about 0.5 to 1 μL.

The surface of the sample plate 40 is covered with a hydrophobic film 42. A hydrophilic region 44 is formed on a part of the surface of the film 42. In the plan view of the film 42, the region 44 has a circular shape. The diameter of the region 44 is about 600 to 900 μm. By applying the sample solution on the region 44 of the film 42, the droplet Dp can be formed on the sample plate 40.

As the solvent in the droplet Dp evaporates over time, the droplet Dp is concentrated in the central portion of the region 44, as shown in FIG. 8B. Further volatilization of the solvent eventually results in crystals of the sample solution, as shown in FIG. 8 (C). The high concentration of crystals makes it possible to analyze trace amounts of sample solutions. In particular, a small amount of sample is targeted in the analysis of biopolymers, and a method for obtaining high-concentration crystals is indispensable for MALDI-TOF-MS.

However, the above-mentioned general pretreatment method has the following problems.
First, there is a problem that it is difficult to increase the concentration ratio of the sample solution. Droplets Dp are formed in the region 44 of the film 42 due to the surface tension of the solvent with respect to the region 44. The liquid volume of the droplet Dp is determined by the area of the region 44. The larger the area of the region 44, the more the liquid amount of the droplet Dp can be increased. However, in the process of evaporating the solvent in the sample solution, the solvent in the contact portion with the region 44 remains without evaporating, so that the concentration ratio of the sample solution is limited contrary to the area of the region 44. If the area of the region 44 is reduced, the contact portion of the sample solution is also reduced, so that the amount of the solvent remaining in the region 44 can be reduced. However, if the area of the region 44 is reduced, the amount of the droplet Dp that can be formed on the region 44 itself decreases, so that it becomes difficult to obtain a sample in an amount sufficient for analysis.

Second, there is a problem that the droplet Dp cannot be formed on the sample plate 40 depending on the type of sample. In particular, when the oil content of the sample is large, the sample solution cannot be applied to the film 42, which makes it difficult to form droplets Dp.

Thirdly, there is a problem that the sample solution is lost. When the sample solution is applied to the region 44, a part of the sample solution may adhere to the hydrophobic region surrounding the outer periphery of the region 44. In this case, loss of the sample solution occurs, and it becomes difficult to obtain an appropriate concentration effect.

On the other hand, in the pretreatment method according to the first embodiment, the droplet Dp can be held in the air by suspending the droplet Dp of the sample solution using ultrasonic waves. According to this, since it is not necessary to form the droplet Dp on the film 42 of the sample plate 40 as in the comparative example, the solvent does not remain in the region 44. As a result, the concentration ratio of the sample solution can be increased. Further, since the sample solution is not applied to the membrane 42, the type of the sample is not selected and the sample solution is not lost.

In the pretreatment method according to the first embodiment, the amount of the sample solution that can be held as a droplet Dp at the position P1 of the node of the acoustic standing wave is about 10 μL, although it varies depending on the specific gravity of the solvent and the surface tension. Can be done. According to this, since the amount of the sample solution (0.5 to 1 μL) that can be applied to the membrane 42 can be increased to about 10 times by a general pretreatment method, it is possible to obtain crystals having a higher concentration. Become. By obtaining high-concentration crystals, the S / N ratio is improved in the mass spectrum, so that the reliability of each peak can be improved. As a result, the sensitivity of the trace analysis can be increased and the reproducibility of the analysis result can be improved.

[Embodiment 2]
FIG. 9 is a diagram for explaining a processing procedure of the pretreatment step according to the second embodiment. FIG. 9 shows a step of concentrating the sample solution (S12) and a step of placing the sample solution on the sample plate (S13).

First, as shown in FIG. 9A, the sample solution is introduced into the microsyringe 50, and a droplet Dp of the sample solution is formed at the tip thereof. The droplet Dp has a liquid volume of about 10 μL. It should be noted that the formation of the droplet Dp can be automatically performed by using a mechanism having a robot arm to which the microsyringe 50 is attached, instead of the manual operation by the user.

Next, as shown in FIG. 9B, hold the tip of the microsyringe 50 in a state of facing downward in the vertical direction. The droplet Dp is held at the tip of the microsyringe 50 by its own weight. The droplet Dp is concentrated by gradually evaporating the solvent from the droplet Dp held in the air.

Next, as shown in FIG. 9C, the concentrated droplet Dp is taken out from the tip portion of the microsyringe 50. When the user takes out the droplet Dp from the tip of the microsyringe 50 using the micropipette 30, the user proceeds to the next step (S13) and places the taken out droplet Dp on the sample plate 40 (FIG. 9 (D)). reference). The droplet Dp can be taken out automatically by using the above mechanism instead of the manual operation by the user.

Returning to FIG. 3, in the step (S14) of mixing the sample solution and the matrix, the matrix is mixed with the droplet Dp of the sample solution placed on the sample plate. In the step (S14), the mixed solution of the sample solution and the matrix placed on the sample plate 40 is dried to volatilize the solvent in the mixed solution and crystallize it.

FIG. 10 is a diagram showing an example of a mass spectrum created in the analysis step (S20). FIG. 10A is an example of a mass spectrum created by mass spectrometry of a sample by a general pretreatment method (see FIG. 8). FIG. 10B is an example of a mass spectrum created by mass spectrometry of a sample by the pretreatment method (see FIG. 9) according to the second embodiment.

In each of FIGS. 10 (A) and 10 (B), three mass spectra having different concentrations of the samples contained in the sample solution are shown superimposed. The sample solution is a peptide (ACTH18-39). In each figure, the concentrations of the sample solutions of the three mass spectra are 2.5 fmol / μL, 250amol / μL, and 25amol / μL in order from the top.

As shown in FIG. 10A, the detection limit of the analyzer 20 using a general pretreatment method is 250 amol / μL. The detection limit is the minimum amount of a substance that can distinguish between the system noise (blank value) in the absence of the substance and the signal of the substance. With a general pretreatment method, it is impossible to detect a peak in the mass spectrum at 25 amol / μL, which is 1/10 of the detection limit.

On the other hand, as shown in FIG. 10B, in the pretreatment method according to the second embodiment, a peak can be detected in the mass spectrum at 25 amol / μL. This indicates that in the pretreatment method according to the second embodiment, the concentration ratio of the sample solution is increased to about 10 times as compared with the general pretreatment method. In the pretreatment method according to the second embodiment, the S / N ratio in the mass spectrum is improved, so that the sensitivity of the trace analysis can be increased. Furthermore, the reproducibility of the analysis result can be improved.

[Other configuration examples]
In the above-described first and second embodiments, the sample pretreatment method when the analyzer 20 is MALDI-TOF-MS has been described, but the sample pretreatment method may be changed according to the type of the analyzer 20. Can be done.

For example, when the MALDI method is not used for ionizing the sample in mass spectrometry, the process of mixing the matrix with the concentrated sample solution and the process of crystallizing the mixed solution (S14, S15 in FIG. 3) may be omitted. can.

Alternatively, when the analyzer 20 is a Raman spectrophotometer, the pretreatment method can be configured such that the concentrated sample solution is placed on a sample plate and crystallized and introduced into the analyzer 20.

Alternatively, when the analyzer 20 is FT-IR, it may be configured to irradiate infrared light with the droplet Dp of the concentrated sample solution suspended and measure the amount of transmitted or reflected light. can.

Further, in the above-described first and second embodiments, an example in which the user visually determines the concentration rate of the droplet Dp has been described, but as another configuration example, the configuration shown in FIG. 11 is used to determine the concentration of the droplet Dp. The enrichment rate may be controlled.

The ultrasonic floating device 12A according to another configuration example shown in FIG. 11 is a step of concentrating the sample solution instead of the cameras 13 and 14 (see FIG. 4) that indicate the position of the node P1 of the acoustic standing wave W (see FIG. 4). A camera 113 for photographing the droplet Dp over time (continuously or intermittently) in S12) is provided. As a matter of course, the camera 113 of FIG. 11 may have a purpose of indicating the position of the node P1 as in the

cameras

13 and 14 of FIG. Further, although not shown, in this configuration example, the pretreatment device 10 is provided with an input unit that receives an input of a desired concentration rate for the sample from the user.

The camera 113 is connected to a control device 114 having a calculation unit and a storage unit. The arithmetic unit of the control device 114 can execute a program for recognizing the droplet Dp from the image taken by the camera 113 and acquiring the dimension (and the corresponding volume) thereof. When calculating the volume from the dimensions of the droplet Dp, the shape of the droplet Dp may be regarded as a spherical shape. Further, the calculation unit can execute a program for calculating the ratio (concentration rate) of the initial droplet Dp to the volume after the lapse of time.

The camera 113 captures the droplet Dp after the droplet Dp is held in the node P1 (preferably immediately after), and acquires the droplet image. The control device 114 acquires the volume of the droplet Dp from the droplet image in the calculation unit and stores it in the storage unit. After that, the camera 113 continuously (for example, as a moving image) or intermittently captures the droplet Dp. The volume of the droplet Dp is continuously (or intermittently) calculated and stored in the control device 114. Then, the control device 114 may notify the user when the enrichment rate calculated at the same time as the volume reaches the user's desired value. As a method of notification, a display unit may be provided on the pretreatment device 10 to indicate that the enrichment rate has reached a desired value by the user.

As a further modification of the configuration example of FIG. 11, the time change of the enrichment rate may be obtained, the expected time until the enrichment rate reaches the user's desired value, and displayed on the display unit. Alternatively, a straight line or a curve indicating the time change of the concentration rate may be displayed on the display unit.

[Aspect]
It will be understood by those skilled in the art that the plurality of exemplary embodiments described above are specific examples of the following embodiments.

(Clause 1) The sample pretreatment method according to one embodiment includes a step of generating an acoustic standing wave, a step of introducing a liquid sample into the acoustic standing wave, and a liquid sample at the position of a node of the acoustic standing wave. It is provided with a step of concentrating the liquid sample by holding the above.

According to the sample pretreatment method according to one embodiment, since the liquid sample can be concentrated in a state where the droplets are held in the air in a non-contact manner, a general pretreatment method for forming the droplets on the sample plate can be performed. As such, the solvent does not remain on the surface of the sample plate. This makes it possible to increase the concentration ratio of the liquid sample. Moreover, since the liquid sample is not applied to the sample plate, any type of sample can be used. Therefore, since crystals having a high concentration can be obtained regardless of the type of sample, it is possible to analyze a small amount of sample with high sensitivity.

(2) The sample pretreatment method according to paragraph 1 further comprises a step of placing the concentrated liquid sample on the sample plate.

According to this, a sample concentrated to a higher concentration than a general pretreatment method for concentrating a liquid sample on a sample plate can be placed on the sample plate.

(Clause 3) The sample pretreatment method according to paragraph 2 is a step of mixing a matrix with a liquid sample placed on a sample plate and a step of drying and crystallizing a mixed solution of the liquid sample and the matrix. And further prepare.

According to this, since high-concentration crystals can be ionized by the MALDI method, the analysis sensitivity of the ionized sample can be improved.

(Clause 4) The sample pretreatment method according to paragraph 1 relates to a step of acquiring an image of a liquid sample over time and a degree of concentration of the liquid sample based on the acquired images of a plurality of liquid samples. Further provided with steps to obtain information.

According to this, the degree of concentration of the liquid sample can be controlled to a value desired by the user by using the acquired information.

(Section 5) The analysis method according to one aspect includes the sample pretreatment method according to paragraph 1 and the step of introducing the sample into the analyzer.

According to the analysis method according to one aspect, the S / N ratio of the signal acquired by the analyzer can be improved, so that the sensitivity of the analysis can be increased.

(Section 6) The analysis method according to one embodiment includes a step of holding a liquid sample at the tip of an elongated holding member to concentrate the liquid sample, a step of placing the concentrated liquid sample on a sample plate, and a liquid. It includes a step of introducing the sample into the analyzer.

According to the analysis method according to one embodiment, the liquid sample can be concentrated while the droplets are held in the air, so that the solvent can be used as in a general pretreatment method for forming droplets on a sample plate. Does not remain on the surface of the sample plate. This makes it possible to increase the concentration ratio of the liquid sample. Moreover, since the liquid sample is not applied to the sample plate, any type of sample can be used. Therefore, since crystals having a high concentration can be obtained regardless of the type of sample, it is possible to analyze a small amount of sample with high sensitivity.

(Section 7) The sample pretreatment device according to one embodiment includes a device for generating an acoustic standing wave, a means for introducing a liquid sample into the acoustic standing wave, and a liquid sample at the position of a node of the acoustic standing wave. It is provided with a means for concentrating a liquid sample by holding the above.

According to the sample pretreatment apparatus according to one embodiment, the liquid sample can be concentrated while the droplets are held in the air in a non-contact manner, so that the concentration ratio of the liquid sample can be increased regardless of the type of sample. Can be done. As a result, high-concentration crystals can be obtained regardless of the type of sample, so that a small amount of sample can be analyzed with high sensitivity.

(Section 8) The sample pretreatment apparatus according to paragraph 7 further includes means for placing the concentrated liquid sample on the sample plate.

(Section 9) The sample pretreatment device according to paragraph 7 is further equipped with a camera for indicating the position of the node of the acoustic standing wave.

According to this, the liquid sample can be introduced exactly at the position of the node of the acoustic standing wave.
(Item 10) The sample pretreatment apparatus according to item 7 concentrates a liquid sample based on a camera that acquires images of the liquid sample over time and images of a plurality of liquid samples acquired by the cameras. It is further equipped with a control device for acquiring information on the degree.

(Item 11) The sample pretreatment apparatus according to item 7 is a means for mixing a matrix with a liquid sample placed on a sample plate and a means for drying and crystallizing a mixed solution of the liquid sample and the matrix. And further prepare.

(Section 12) The analysis system according to one aspect includes the sample pretreatment device according to the seventh paragraph and the analysis device for analyzing the sample introduced from the pretreatment device.

According to the analysis system according to one aspect, the S / N ratio of the signal acquired by the analyzer can be improved, so that the sensitivity of the analysis can be increased.

(Item 13) In the analysis system according to one embodiment, a means for holding a liquid sample at the tip of an elongated holding member to concentrate the liquid sample, a means for placing the concentrated liquid sample on a sample plate, and a liquid. It is provided with a means for introducing the sample into the analyzer.

According to the analysis system according to one embodiment, the liquid sample can be concentrated while the droplets are held in the air, so that the solvent can be used as in a general pretreatment method for forming droplets on a sample plate. Does not remain on the surface of the sample plate. This makes it possible to increase the concentration ratio of the liquid sample. Moreover, since the liquid sample is not applied to the sample plate, any type of sample can be used. Therefore, since crystals having a high concentration can be obtained regardless of the type of sample, it is possible to analyze a small amount of sample with high sensitivity.

It should be noted that, with respect to the above-described embodiments and modifications, it is initially filing that the configurations described in the embodiments are appropriately combined within a range that does not cause any inconvenience or contradiction, including combinations not mentioned in the specification. Scheduled from.

The embodiments disclosed this time should be considered to be exemplary in all respects and not restrictive. The scope of the present invention is shown by the scope of claims rather than the above description, and is intended to include all modifications within the meaning and scope of the claims.

10 pretreatment device, 12 ultrasonic floating device, 13, 14, 113 camera, 20 analyzer, 22 sample introduction section, 24 measurement section, 26 processing section, 30 micropipette, 40 sample plate, 42 membrane, 44 region, 50. Microsyringe, 100 analysis system, 114 control device, 120 ultrasonic oscillator, 122 horn, 124 reflector, 126 oscillator, 128 commercial power supply, 240 ionization unit, 242 laser light source, 250 mass separator, 252 flight tube, 260 detection Vessel, S sample, Dp droplet, W acoustic standing wave.

Claims

Steps to generate acoustic standing waves and
The step of introducing a liquid sample into the acoustic standing wave and
A sample pretreatment method comprising a step of concentrating the liquid sample by holding the liquid sample at a node of the acoustic standing wave.
The sample pretreatment method according to claim 1, further comprising a step of placing the concentrated liquid sample on a sample plate.
The step of mixing the matrix with the liquid sample placed on the sample plate,
The sample pretreatment method according to claim 2, further comprising a step of drying and crystallizing the liquid sample and the mixed solution of the matrix.
The step of acquiring an image of the liquid sample over time and
The sample pretreatment method according to claim 1, further comprising a step of acquiring information regarding the degree of concentration of the liquid sample based on the acquired images of the liquid sample.
The sample pretreatment method according to claim 1 and
An analytical method comprising the step of introducing the sample into an analyzer.
A step of holding a liquid sample at the tip of an elongated holding member and concentrating the liquid sample,
The step of placing the concentrated liquid sample on the sample plate and
An analytical method comprising the step of introducing the liquid sample into an analyzer.
A device that generates an acoustic standing wave,
A means for introducing a liquid sample into the acoustic standing wave,
A sample pretreatment apparatus comprising a means for concentrating the liquid sample by holding the liquid sample at the position of the node of the acoustic standing wave.
The sample pretreatment apparatus according to claim 7, further comprising means for placing the concentrated liquid sample on a sample plate.
The sample pretreatment apparatus according to claim 7, further comprising a camera for indicating the position of the node of the acoustic standing wave.
A camera that acquires images of the liquid sample over time,
The sample pretreatment apparatus according to claim 7, further comprising a control device for acquiring information regarding the degree of concentration of the liquid sample based on the images of the plurality of liquid samples acquired by the camera.
A means for mixing a matrix with the liquid sample placed on the sample plate, and
The sample pretreatment apparatus according to claim 8, further comprising a means for drying and crystallizing the liquid sample and the mixed solution of the matrix.
The sample pretreatment apparatus according to claim 7,
An analysis system including an analysis device for analyzing the sample introduced from the pretreatment device.
A means for holding a liquid sample at the tip of an elongated holding member and concentrating the liquid sample,
A means for placing the concentrated liquid sample on a sample plate, and
An analytical system comprising means for introducing the liquid sample into an analyzer.