JP2007033388A - Method and apparatus for acquiring information - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique excellent in both sensitivity and reproducibility in an information acquisition method for acquiring the mass numbers of molecules contained in a sample solution. <P>SOLUTION: The information acquisition method for acquiring information on the mass numbers of molecules present in a liquid includes: a first process for discharging the liquid as droplets; a second process for acquiring ions of molecules present in the liquid by receiving the discharged droplets at a liquid pool part, making them flow through an inlet channel, electrostatically spraying them from a sharp-pointed part to form electrically charged finer liquid particulates and by the transpiration of the liquid from liquid particulates; and a third process for acquiring information on the mass numbers of the ions by mass spectrometry. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an information acquisition method and an information acquisition apparatus for acquiring information about a reaction when a mass number of molecules present in a solution and a plurality of types of solutions are mixed.

  The principle of electrospray ionization is to first form a charged droplet by electrostatically spraying the sample solution from the tip of a capillary to which a high potential is applied, and then using this heat and inert gas to produce the charged droplet. By removing the solvent, the charge is transferred to the sample molecules and ionized.

  The molecules ionized in this way are guided to a mass spectrometer and are subjected to mass spectrometry.

  Electrospray ionization is widely used as an ionization method from low molecular weight compounds to biopolymers.

  In particular, this ionization method does not apply high heat and does not collide high-energy particles, so it has the characteristic of ionizing substances under mild conditions, so it destroys biopolymers such as peptides, proteins, and nucleic acids. The feature is that multivalent ions can be generated without any problems.

  Since the solution sample is the target, it is used as an ionization method when a sample solution is directly introduced using a syringe pump, or as an ionization method of LC / MS combined with liquid chromatography or CE / MS combined with capillary electrophoresis. ing.

  For general electrospray ionization, a metal capillary having an inner diameter of about 100 to 120 μm or a silica capillary having an inner diameter of 20 to 100 μm is used.

  Although the flow rate of the sample solution varies depending on the inner diameter, it is practically 100 to 10,000 nl / min. It is.

  In the 1990s, 30 to 500 nl / min. Nano-electrospray ionization capillaries (nano-electrospray ionization method) that can be ionized at extremely low flow rates have appeared (Patent Document 1).

  This is made of glass or fused silica having an inner diameter of 5 to 50 μm coated with a metal thin film or a conductive polymer thin film, and has a flow rate of 30 to 3000 nl / min. It was possible to ionize the solution sample, and the solution sample amount could be reduced.

  A flow rate of 100 to 300 nl / min. Using a column with an inner diameter of 50 to 100 μm. By combining this nano-LC with this nano-electrospray ionization method, ultra-low flow nano-LC / MS can be performed.

  Nano LC / MS is widely used in the biotechnology field such as proteomics for analyzing extremely small amount of protein in the living body, because the sample separation and concentration effect by nano LC can reduce the sample amount and increase the sensitivity. (Non-Patent Document 1).

  In addition, by reducing the flow rate, the efficiency of ion incorporation into the mass spectrometer can be improved.

In general, the uptake efficiency of ions generated by electrospray ionization is extremely low, 10 ⁻⁶ to 10 ⁻¹⁰ , but can be improved to about 10 ⁻² by reducing the flow rate of the sample solution (Non-patent Document 2).

  As a sample introduction method, each sample to be measured is placed in a container and introduced using an autosampler or the like.

For example, in order to investigate what kind of reaction occurs when a plurality of samples are mixed, a sample obtained by reacting the sample in advance is prepared in a container, and measurement is performed by introducing the sample.
US Pat. No. 5,788,166 Naoyuki Yamada; Mass. Spectrom. Soc. Jpn. , 2000, 48 (3), 187-192 M.M. Wilm et al. Anal. Chem. 1996, 68, 1-8

  In this way, the concentration effect by nano LC and the improvement of ion uptake efficiency have improved the sensitivity and the required amount of sample, but it can be said that the reproducibility is good with the sample introduction using the conventional capillary pump. First, it takes a lot of labor and time to analyze a large number of extremely small samples.

  Also, in terms of sensitivity, it is difficult to obtain a stable flow rate in the region of several tens of nl / min or less, and there is a limit to the improvement of ion uptake efficiency.

  Furthermore, when introducing a sample, it is necessary to put it in a container in advance for each sample to be measured. In order to investigate what kind of reaction occurs when multiple types of samples are mixed, the sample must be reacted in advance. It was necessary to prepare the container in a container.

  Further, since the amount of the sample is extremely small, there is a problem that the influence of the measurement error at the time of sample contamination and sample preparation is large.

  The present invention solves the above-mentioned problems, and can measure a very small amount of sample in a short time with good reproducibility, and the flow rate is 1 nl / min. A method for further improving sensitivity is provided by the following.

  In addition, a method is provided in which a plurality of samples are mixed with little influence of contamination and measurement error, and the reaction due to the mixing can be easily investigated.

  In order to solve the above problems, the present invention provides an information acquisition method for obtaining information on the mass number of molecules present in a liquid, the first step of discharging the liquid as droplets, The discharged liquid droplets are received by the liquid reservoir, and flowed through the introduction passage, and electrostatic spraying is performed from the sharpened portion to form a finer liquid particle having a charge. A second step of obtaining ions of molecules existing in the liquid; and a third step of obtaining information on the mass number of the ions by mass spectrometry.

  The present invention also relates to an information acquisition apparatus, an inkjet nozzle that supplies a sample solution, a droplet reservoir that receives droplets ejected from the inkjet nozzle, and a voltage applied to the droplets in the droplet reservoir. An introduction passage that leads to a portion to which a voltage is applied, a sharp point that electrostatically sprays a droplet guided to a portion to which a voltage is applied from the introduction passage, into a finer liquid droplet, and heat is applied to the liquid droplet It comprises a heater, an ion intake port for taking in ions of molecules contained in the liquid, and a mass spectrometer for separating the taken-in ions based on mass.

  In this way, the supply of the solution to the ion source is performed with a very small amount of droplets that are not easily affected by contamination during sample preparation, such as the ink jet method, and has excellent volumetric capacity. By performing electrospraying at a stable flow rate by electrospraying, a very small amount of sample can be measured in a short time with good reproducibility, and the flow rate is 1 nl / min. The following can be done.

  Thereby, further sensitivity improvement can be realized.

  In addition, since the inkjet method has high landing accuracy, the sample can be mixed by controlling a plurality of types of sample droplets, and the reaction that occurs when mixing can be easily investigated.

  In addition, by installing a holding material that can be used as liquid chromatography in the solution transport passage in the ion source, components in the liquid can be separated and measured.

  ADVANTAGE OF THE INVENTION According to this invention, in the information acquisition method which acquires the mass number of the molecule | numerator contained in a sample solution, it is excellent in a sensitivity and the method of good reproducibility is provided.

  In addition, a method is provided that can easily investigate the reactions of a plurality of types of use solutions.

  Hereinafter, the present invention will be described in more detail.

  FIG. 3 is a schematic diagram showing an embodiment of the information acquisition apparatus of the present invention.

  In FIG. 3, 10 is an inkjet nozzle that supplies a sample solution, 20 is an electrospray that mists the sample solution by electrostatic spraying, 25 is an ion intake port of the mass spectrometer, and 40 is a mass spectrometer body.

  The sample solution discharged from the inkjet nozzle 10 as a fixed amount of droplets lands on the liquid reservoir 21 at the top of the electrospray 20.

  The landed sample solution is sent to the sharpened tip 23 by the capillary tube 22, charged by the sharpened portion 23, and sprayed into the air as further fine droplets (mist).

  As the mist floats in the air, the liquid evaporates, and the molecules contained in the mist are charged and enter the ion intake port 25.

  The captured ions are separated and detected by the mass analyzer 40.

  Hereinafter, it explains in detail according to a process.

  First, the first step of discharging the liquid as droplets will be described.

  In order to stably eject a stable mist from the electrospray, it is necessary to flow the sample solution through the capillary 22 at a constant flow rate for a fixed time. For this purpose, a certain amount of sample solution is supplied to the liquid reservoir 21. The ink jet method is optimal because it can be formed into droplets while the volume of the sample solution is controlled and does not damage the sample solution.

  The sample solution supply to the ink jet nozzle can be performed from the solution supply tank when there is a large amount of solution.

  When the amount of the solution is extremely small, it is possible to collect the solution by capillary action or the like and use the capillary as a solution supply tank.

  Furthermore, by allowing time before ejection, the solvent evaporates from the gas-liquid interface of the solvent, thereby obtaining a sample concentration effect.

  In addition, since the ink jet method has high landing accuracy, the sample can be mixed by controlling a plurality of types of sample droplets.

  Thereby, the reaction product generated upon mixing can be easily prepared.

  Next, a second step of applying a high voltage to the droplet to perform electrostatic spraying to form a finer droplet and obtaining molecular ions present in the droplet will be described.

  Electrostatic spraying is a phenomenon in which droplets are dispersed into very fine droplets of about 10 nm by Coulomb force.

  In the present invention, the following device has been devised as a configuration for causing this phenomenon to occur in droplets ejected by inkjet or the like.

  That is, the droplet reservoir 21 that can receive the ejected droplets, and the liquid temporarily held in the droplet reservoir 21 are sequentially transported to the tip sharpened portion 23 to which a high voltage is applied by capillary action. The configuration has an introduction passage 22.

  The introduction passage 22 has a thin tube diameter corresponding to the volume of the droplet so that a small amount of the droplet in the liquid reservoir 21 flows at a constant flow rate for a certain period of time.

  In the present embodiment, the capillary has a diameter of 2 μm and a length of 100 μm.

  At this time, since the volume of the capillary is about 0.4 pl, if the sample solution in the droplet reservoir 21 is about 10 pl, the sample solution can be sequentially introduced into the capillary at a stable flow rate, and is constant for a certain time. Capillary flow can be made.

  At the tip of the sharpened portion 23, an electrostatic spray phenomenon occurs due to the applied high voltage, whereby the sample solution becomes a charged fine droplet.

  Furthermore, in order to obtain molecular ions present in the sprayed droplets, a heat source that desorbs excess solvent by applying heat is provided as a constituent element, whereby ions in the solution can be finally obtained.

  Moreover, the component in a liquid can be isolate | separated and measured by installing the holding material which can be used as a liquid chromatography in the channel | path of solution transportation.

  For example, a component separation effect can be obtained by chemically modifying an octadecylsilyl group on the inner wall of a passage having a diameter of about 2 μm.

  In particular, this is effective for sample desalting.

  In electrospray ionization, if a salt is present in a sample solution, its ionization efficiency is greatly reduced, so this configuration having a desalting effect is effective.

  Next, the third step for obtaining information on the mass number of the ions by mass spectrometry will be described.

  The ions obtained by the second step exist under atmospheric pressure.

  In general, mass spectrometry is performed by examining the behavior of ions in an electromagnetic field under high vacuum.

  For this reason, the high-vacuum mass spectrometer is loaded by, for example, differential evacuation.

  At this time, it is desirable to induce ions by the extraction electrode in order to increase the ion uptake efficiency.

  In general, when the flow rate of the sample solution to be ionized is large, the spray spread direction and the ion take-in direction are set so as not to be uniaxial so as not to contaminate the inside of the analyzer. Therefore, it is easy to place the spray spreading direction and the ion uptake direction on one axis so as to be effective for improving the uptake efficiency of ions.

  Furthermore, it is possible to further improve the ion uptake efficiency by converging the ions with a quadrupole lens or the like.

  The ions taken in this way can be measured for mass by techniques such as time-of-flight mass spectrometry, double-focusing mass spectrometry, quadrupole mass spectrometry, and Fourier transform ion cyclotron mass spectrometry. it can.

  Hereinafter, the present invention will be described more specifically with reference to examples.

  The specific example shown below is an example of the best embodiment according to the present invention, but the present invention is not limited to such specific form.

(1) Production of Sample Supply Device by Inkjet Inkjet heads 10, 10 ′ (droplet capacity: 30 pl) are prepared, and a system capable of supplying sample droplets is produced. In this system, two types of solutions can be discharged to the same position at once (FIG. 1).
(2) Production of Electrospray Ion Source 20 for Inkjet Droplets An anisotropic etching is performed on a 250 μm thick Si substrate to dig a quadrangular pyramid depression (bottom size: 200 μm × 200 μm, depth 140 μm). On the opposite surface, a quadrangular pyramidal projection (bottom size: 200 μm × 200 μm, height 140 μm) is formed by anisotropic etching.

  A passage having a diameter of 2 μm is formed so as to connect the depression and the apex of the protrusion.

Gold is vapor-deposited on the surface on the protrusion side. In addition, the depression forms an SiO ₂ layer by oxidation of the Si substrate and forms ODS (octadecylsilyl) groups by silane coupling treatment (FIG. 2).
(3) Manufacture of mass spectrometer equipped with sample supply device by inkjet and electrospray ion source for inkjet droplets Time-of-flight mass spectrometer (Q-Tof Ultimate API manufactured by Waters) 40 is the above (1), (2 The sample supply devices 10 and 10 'by inkjet and the electrospray 20 for inkjet droplets and the ceramic heater 30 are installed (FIG. 3).
(4) Analysis of DNA hybridization using a mass spectrometer equipped with an inkjet sample supply device and an electrospray ion source for inkjet droplets Solution 1 (500 μM synthetic oligo DNA) using the device described in (3) above Solution (Funakoshi Co., Ltd.) 12mer: AGC TAG CTA GCT Solvent: Express Hyb hybridization solution (Nippon Becton Dickinson Co., Ltd.) and Solution 2 (500 μM synthetic oligo DNA (Funakoshi Co., Ltd.) 12mer: TCG ATC GAT CGA Solvent: Express Hyb hybridization solution (Nippon Becton Dickinson Co., Ltd.) is installed on each of the inkjet heads 10 and 10 'and discharged simultaneously.

  As a result, a mass number peak indicating that the DNA has hybridized can be obtained.

  The present invention can be effectively used for obtaining information on the mass number of molecules contained in a plurality of types of samples.

It is a figure which shows the supply system of the sample solution using an inkjet. It is a figure which shows the electrospray ion source corresponding to an inkjet droplet. It is a figure which shows the mass spectrometer provided with the sample supply by inkjet and the electrospray ion source for inkjet droplets.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10, 10 'Inkjet head 20 Electrospray 21 Liquid reservoir part 22 Capillary 23 Sharp part 25 Ion intake port 30 Ceramic heater 40 Mass spectrometer

Claims (9)

An information acquisition method for obtaining information on the mass number of molecules present in a liquid,
A first step of discharging the liquid as droplets;
The discharged liquid droplets are received by the liquid reservoir, flowed through the introduction passage, and electrostatic spraying is performed from the sharpened point to form a finer liquid particle having a charge. From the liquid particle, the liquid is evaporated. A second step of obtaining ions of molecules present in the liquid;
A third step of obtaining information on the mass number of the ions by mass spectrometry;
An information acquisition method comprising: The information acquisition method according to claim 1, wherein the first step is a step of discharging droplets from an inkjet nozzle. The information acquisition method according to claim 1, wherein the second step includes a step of heating the liquid droplet. The first step is a step of sequentially discharging a plurality of types of droplets from a plurality of inkjet nozzles, and the third step is sequentially information on the mass number of molecules contained in each of the droplets. The information acquisition method according to claim 2, wherein the information acquisition method is an acquisition step. The first step is a step of discharging a plurality of types of droplets from a plurality of inkjet nozzles, and the second step includes a step of mixing and reacting the plurality of droplets, 3. The information acquisition method according to claim 2, wherein the step is a step of acquiring information on the mass number of the product of the reaction. The plurality of types of droplets are a combination of a solution in which DNA is dissolved and a solution capable of labeling a specific base sequence in mass information, and the identification is performed by mass analysis of molecules generated by the reaction of each solution. The information acquisition method according to claim 5, wherein information on the presence of the base sequence is obtained. The plurality of types of droplets is a combination of a solution in which a protein is dissolved and a solution capable of labeling a specific protein in mass information, and the specific analysis is performed by mass analysis of molecules generated by the reaction of each solution. The information acquisition method according to claim 5, wherein information on the presence of the protein is obtained. An inkjet nozzle for supplying a sample solution;
A droplet reservoir for receiving droplets ejected from the inkjet nozzle;
An introduction passage for guiding the droplet received in the droplet reservoir to a site to which a voltage is applied;
A sharpened portion for electrostatically spraying the droplet introduced into the site to which the voltage is applied from the introduction passage into a finer liquid particle;
A heater for applying heat to the liquid particles;
An ion intake port for taking in ions of molecules contained in the liquid;
A mass spectrometer for separating the incorporated ions based on mass;
An information acquisition apparatus comprising: 9. The information acquisition apparatus according to claim 8, wherein the introduction passage is a passage that separates components in the liquid by liquid chromatography.

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