JPWO2004051252A1 - Separation apparatus and mass spectrometry system using the same - Google Patents

Abstract

The sample (112) is introduced into the sample introduction channel (114) and guided to the electrophoresis lane (120) through the fine channel provided in the partition wall (118). In the electrophoresis lane (120), an electrode (116a) and an electrode (116b) are arranged to face each other. The sample (112) is introduced from the side with respect to the direction of the electric field constituted by these electrodes. Since the sample (112) includes an isoelectric electrolyte, a pH gradient is formed in the electrophoresis lane (120) due to the influence of the electric field. For this reason, the sample introduced from the fine channel of the partition wall (118) converges into a band in the electrophoresis lane (120) according to the isoelectric point of each component, as shown. The converged band component is quickly taken into the electrophoresis lane (120).

Description

  The present invention relates to a separation apparatus for separating a sample by an isoelectric point or the like, and a mass spectrometry system using the same.

Proteomics is attracting attention as a research method that plays a part in the post-genomic era. In proteomics research, proteins and the like are finally identified by mass spectrometry or the like, but in the previous stage, sample separation and pretreatment are performed to enable mass spectrometry and the like. Conventionally, two-dimensional electrophoresis has been widely used as such a sample separation technique. In two-dimensional electrophoresis, amphoteric electrolytes such as peptides and proteins are separated at their isoelectric points, and further separated according to molecular weight.
However, this separation method usually takes time so that it takes a whole day and night, and the recovery rate of the sample is low, so that only a relatively small amount of sample can be obtained for mass spectrometry and the like.
On the other hand, in recent years, microchemical analysis (μ-TAS) in which chemical operations such as sample pretreatment, reaction, separation, and detection are performed on a microchip is rapidly developing. According to the separation / analysis method using a microchip, a small amount of sample is used, and the environmental load is small and highly sensitive analysis is possible. It is also possible to greatly reduce the time required for separation.
Patent Document 1 describes an apparatus that realizes capillary electrophoresis with a microchip having a structure in which a groove or a reservoir is provided on a substrate.
Patent Document 1 Japanese Patent Application Laid-Open No. 2002-207031 Patent Document 2 describes a microchip type electrophoresis apparatus that separates and analyzes peptides, proteins, and the like at their isoelectric points.
Patent Document 2 Japanese Patent Application Laid-Open No. 2000-55879 Hereinafter, an apparatus described in Patent Document 2 will be described with reference to FIG. In this apparatus, a microchip 18 in which a separation channel 14 and a sample introduction channel 16 intersect with each other is provided inside a glass substrate. On one surface of the microchip 18, holes reaching the separation channel 14 or the sample introduction channel 16 are formed at positions corresponding to both ends of the separation channel 14 and both ends of the sample introduction channel 16. An electrophoresis power supply device 20 that applies an electrophoresis voltage to both ends of the separation channel 14 and to both ends of the sample introduction channel 16 is provided.
The light irradiation means 26 includes a light source 22 and a spectroscope 24 and irradiates a predetermined range of the separation channel 14 with light. On the side opposite to the light irradiation means 26, light receiving elements that detect light from the separation channel 14 at respective positions are arranged. This light receiving element is constituted by an array type photodetector 28 arranged along the separation channel 14. The array-type photodetector 28 is connected to the CPU 34 via an amplification circuit 30 that amplifies a detection signal and an A / D converter 32 that converts an analog signal into a digital signal. The CPU 34 performs data analysis for sample component identification based on the detection signal. The container 36 stores a marker sample, a sample, and a polybuffer, which are injected into one end of the separation channel 14 and the sample introduction channel 16 by a needle 38.
By using such a microchip separation method, it is possible to perform highly sensitive analysis using a very small amount of sample. However, since these microchips have a small channel size, they can only separate a relatively small amount of material, and have a problem that the amount of sample detected and collected in the separation unit is small. In addition, the above method is a batch-type separation process due to its nature, and has a problem that it is difficult to adopt a method of continuously introducing samples.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a separation apparatus capable of efficiently separating a relatively large amount of sample. Another object of the present invention is to provide a separation apparatus that suppresses diffusion of separation components and enables detection and recovery even with a small amount of substance.
According to the present invention, an isoelectric point separation region where a pH gradient is formed, a pair of electrodes that apply an electric field to the isoelectric point separation region, and a sample introduction unit that introduces a sample into the isoelectric point separation region, Is provided, and the sample is introduced from the side in the electric field application direction through the sample introduction unit.
In this apparatus, a sample to be separated is introduced from the side in the direction of electric field application via an introduction flow path. This is different from the conventional electrophoresis apparatus in which an electrophoretic electric field is applied in the flow path direction from the sample introduction port to the discharge port. According to the present invention, since the sample is introduced as described above, the sample can be continuously introduced. As a result, a relatively large amount of components separated by isoelectric point separation can be obtained. Further, since the diffusion of the separated component is suppressed, even a small amount of substance can be detected and recovered.
In the separation apparatus of the present invention, the sample introduction unit includes a flow channel into which a sample is introduced, and a partition wall interposed between the flow channel and the isoelectric point separation region. And it can be set as the structure by which the some introduction flow path connected to the said isoelectric point separation area | region is formed.
In this apparatus, since the sample is directly introduced from the flow path into the isoelectric point separation region, it is possible to continuously perform the isoelectric point separation while continuously introducing the sample. Thereby, an efficient isoelectric point separation operation can be realized. Since the conventional equipotential point separation apparatus cannot continuously introduce samples, there are certain limits in detection sensitivity and recovery efficiency. On the other hand, since this invention can isolate | separate a sample continuously, such a subject can be solved effectively.
In the separation device of the present invention, the isoelectric point separation region has a rectangular shape, and includes the pair of electrodes along a pair of opposing sides of the rectangular shape, and the one of the remaining sides along the one side. It can be set as the structure which provided the sample introduction part.
In the separation apparatus of the present invention, a configuration may be provided that includes a sample collection unit that communicates with the isoelectric point separation region and collects the sample introduced into the isoelectric point separation region. The sample recovery unit can be configured to be opposed to the sample introduction unit. The sample recovery unit may include a plurality of recovery lanes communicating with the isoelectric point separation region. In addition, the collection ports of the plurality of collection lanes may be densely arranged in a line along the isoelectric point separation region. By adopting such a configuration, the separated sample can be efficiently recovered.
In the separation device according to the present invention, the wall section that defines the plurality of recovery lanes has a cross-section in the extending direction of the isoelectric point separation region, and a sharp shape or curved surface protrudes toward the isoelectric point separation region. It can be set as the structure which has a shape.
According to the present invention, a light irradiating unit that irradiates a predetermined portion of the isoelectric point separation region with light, a light detecting unit that detects light absorption or light emission at the light irradiated portion, and the light absorption or light emission based on the light absorption or light emission. There is provided a separation device further comprising an analysis unit that performs analysis of components contained in a sample. According to this separation apparatus, it is possible to analyze a sample subjected to isoelectric point separation with high sensitivity.
Furthermore, according to the present invention, a molecular weight separation unit that performs component separation based on molecular weight is further provided adjacent to the isoelectric point separation region, and the isoelectric point separation and the molecular weight separation are continuously performed. Is provided.
Furthermore, according to the present invention, a separation means for separating a biological sample according to molecular size or property, a pretreatment means for performing a pretreatment including an enzyme digestion treatment on the sample separated by the separation means, and a pretreatment A mass spectrometry system is provided, comprising: a drying means for drying the sample that has been dried; and a mass spectrometry means for mass-analyzing the dried sample, wherein the separation means includes the microchip described above.
According to this mass spectrometry system, a sample suitable for mass spectrometry can be efficiently adjusted.
As described above, according to the present invention, it is possible to realize a separation apparatus that can separate a relatively large amount of sample on a microchip and can detect and collect even a small amount of substance.

The above-described object and other objects, features, and advantages will become more apparent from the preferred embodiments described below and the accompanying drawings.
FIG. 1 is a diagram showing a configuration of a conventional electrophoresis apparatus.
FIG. 2 is a diagram illustrating a configuration of the isoelectric point separation device according to the embodiment.
FIG. 3 is a diagram illustrating a configuration of the isoelectric point separation device according to the embodiment.
FIG. 4 is a diagram illustrating a configuration of the isoelectric point separation device according to the embodiment.
FIG. 5 is a diagram showing a detailed structure of the sample introduction port and the sample recovery port in FIG.
FIG. 6 is a diagram illustrating a configuration of the separation device according to the embodiment.
FIG. 7 is a schematic configuration diagram of a molecular weight separation unit according to the embodiment.
FIG. 8 is a diagram for explaining the components recovered from the recovery unit in the apparatus of FIG.
FIG. 9 is a diagram illustrating a voltage application method in the molecular weight separation unit and a state of separation of components corresponding thereto.
FIG. 10 is a configuration diagram of a system including the separation apparatus described in the embodiment.
FIG. 11 is a configuration diagram of the separation apparatus described in the embodiment.
FIG. 12 is a schematic diagram showing the configuration of the mass spectrometer.
FIG. 13 is a block diagram of a mass spectrometry system according to the present embodiment.
FIG. 14 is a diagram showing a main part of an apparatus for performing sample concentration on a microchip.

First Embodiment In this embodiment, a sample such as a protein is separated by an isoelectric point. FIG. 2 is a schematic configuration diagram of a system including the separation device. The continuous two-dimensional electrophoresis chip 100 includes a continuous isoelectric point separation unit 110 and a continuous molecular weight separation unit 105. The sample injected into the continuous two-dimensional electrophoresis chip 100 is first introduced into the continuous isoelectric point separation unit 110. In the continuous isoelectric point separation unit 110, the introduced sample is separated into a plurality of bands according to the isoelectric point. The sample separated into each band is then introduced into the continuous molecular weight separation unit 105. A plurality of continuous molecular weight separation units 105 are formed in an array in a direction perpendicular to the migration direction of the continuous isoelectric point separation unit 110. The sample introduced into the continuous molecular weight separation unit 105 is separated according to the molecular weight. Each separated component is collected and then prepared, for example, as a sample for mass spectrometry by a predetermined procedure.
FIG. 3 is a diagram illustrating an example of the configuration of the isoelectric point separation device. The sample 112 is previously mixed with a predetermined buffer. This buffer is a solution that forms a pH gradient when an electric field is applied, and examples thereof include solutions containing Amphorine, Pharmalyte, etc. manufactured by Amercial Biosciences. This solution is introduced into the sample introduction channel 114 and guided to the electrophoresis lane 120 through a fine channel (not shown) provided in the partition wall 118. In the electrophoresis lane 120, an electrode 116a and an electrode 116b are arranged to face each other. The sample 112 is introduced from the side with respect to the direction of the electric field constituted by these electrodes. Since the sample 112 contains an isoelectric electrolyte, a pH gradient is formed in the electrophoresis lane 120 due to the influence of the electric field. For this reason, the sample introduced from the fine flow path (not shown) of the partition wall 118 converges into a band in the electrophoresis lane 120 according to the isoelectric point of each component, as shown. The converged band component is quickly taken into the collection unit 122.
In this embodiment, such a separation device is realized on a microchip. FIG. 4 is a diagram showing the structure of the separation apparatus according to this embodiment. This apparatus is composed of a hollow support base 147 for storing a heater, a buffer flow path, and the like, and a quartz substrate disposed on the support base 147, and a separation portion or the like is formed in a groove shape on the surface of the quartz substrate. The These can be formed using photolithography technology and dry etching technology. Here, a quartz glass substrate is used as a material for the plate-like member disposed on the support base 147, but a silicon substrate having an oxide film on its surface, a plastic substrate, or the like can also be used.
FIG. 5 is a diagram showing a detailed structure of the sample introduction port and the sample recovery port in FIG.
The sample inlet provided in the partition wall 118 has a structure as shown in the enlarged view on the right side in the figure. The sample inlets provided in the partition walls are regularly arranged at intervals B, and the width of the partition walls is A. On the other hand, the sample collection port provided in the collection unit 122 has a cross-sectional shape that protrudes sharply toward the migration lane 120 in the plane in which the migration lane 120 extends, as shown in the enlarged view on the left side of the drawing. ing. The width of the recovery lane is d1, while the width of the recovery port is b. As described above, since the opening has a shape that expands toward the electrophoresis lane 120, the recovery efficiency is improved. Further, by making the cross-sectional shape projecting sharply toward the electrophoresis lane 120 or the shape projecting the curved surface, the sample can be smoothly collected to the adjacent collection lane. By doing so, the liquid flow from the electrophoresis lane 120 toward the recovery unit 122 is not disturbed, and the resolution is improved by taking each component into the lane to be recovered. In addition, although the opening width of each part etc. can be designed arbitrarily, it can be set as the following sizes, for example.
A: 0.1 mm
B: 1mm
a: 0.5 mm
b: 1 mm
d1: 0.5 mm
d2: 0.1 mm
The collected sample can be further separated by another separation device, for example, a molecular weight separation device. Moreover, you may perform the analysis by another analysis method after sample collection.
According to the separation apparatus of this embodiment described above, a relatively large amount of samples can be continuously isoelectrically separated on a microchip. According to this apparatus, even a small amount of substance can be detected and recovered.
In order to concentrate the collected sample, a method using an electric field can be used as follows. FIG. 14 shows the arrangement of the electrodes in the recovery channel. As shown in FIG. 14A, a plurality of electrode plates 334 provided with a gap enough to allow the sample to pass therethrough can be arranged with a distance D in the traveling direction of the sample 112 as D. At this time, the electrode plates 334 are arranged such that the distance D is wider than the width W of the flow path 112, more preferably twice or more the width of the flow path 112. By doing so, it is possible to prevent the phenomenon that the sample cannot enter between the electrode plates 334 due to the influence of the electric lines of force between the electrodes 334. The gap provided in the electrode plate 334 is formed to a size that allows the sample to pass sufficiently. Furthermore, as shown in FIG. 14B, a configuration in which the counter electrode 335 of the electrode 334 is provided between the electrodes 334 charged with a polarity opposite to the charge of the sample may be employed. Accordingly, the sample proceeds toward one of the electrodes 334 on both sides of the counter electrode 335, so that the amount of the sample attached to the electrode 334 can be increased.
Second Embodiment The present embodiment relates to a separation apparatus that separates a sample such as a protein by an isoelectric point and subsequently separates components according to molecular weight.
FIG. 6 is a schematic view of the separation apparatus according to the present embodiment. The right part of the plan view is an isoelectric point separation part having the same structure as that described in the first embodiment. The left side of the plan view is a separation part that separates components according to molecular weight. The components subjected to isoelectric point separation in the electrophoresis lane 120 are guided to the molecular weight separation unit 140 via the mixing unit 130, where they are separated by molecular weight. Looking at the cross-sectional view, the migration lane 120 and the mixing portion 130 are formed as grooves on the surface of the quartz substrate on the support base 147. A heater 132 is provided on the lower surface of the quartz substrate where the mixing portion 130 is formed so that the recovered components can be mixed efficiently. The molecular weight separator 140 is applied with an electric field in the height direction, and is separated according to the molecular weight according to the electric field.
Hereinafter, the details of the operation in each part will be described.
Since isoelectric point separation by the electrophoresis lane 120 has already been described in the first embodiment, the description thereof is omitted here.
The mixing unit 130 plays a role of adjusting components recovered from the electrophoresis lane 120 so as to be suitable for molecular weight electrophoresis. The purpose of mixing is as follows. The first is to neutralize the solution having a biased pH to around the pH of the molecular weight electrophoresis buffer (pH 8.0). Second, the surfactant and the recovered component are reacted to expand and charge the molecules of the recovered component. For this purpose, it is important that a buffer containing an appropriate amount of a surfactant for neutralization is continuously injected in each mixing lane in the mixing section in accordance with the pH.
In order to promote binding between the surfactant and the protein, it is important to heat with a heater. Here, a heater 132 is installed on the lower surface of the mixing unit 130. In addition, SDS etc. are used suitably as surfactant.
Here, the pH of the neutralizing buffer supplied to each flow path of the mixing unit 130 is set as follows. When the number of flow paths is N and the pH range is 2 to 10, isoelectric point-separated samples having different pH by Δ = 8 / N are obtained per flow path. If the first channel is pH 2, the second channel is pH = 2 + Δ. In order to neutralize the pH of each channel, an alkaline neutralizing solution is used when the pH of the channel is acidic, and an acidic neutralizing solution is used when the pH of the channel is alkaline. A Tris-HCl buffer can be used as the neutralizing solution. In this case, Tris is increased when alkaline, and HCl is increased when acidic. Naturally, the neutralizing solution is selected so as to maintain the hydrostatic pressure difference between the isoelectric point recovery unit and the sample introduction unit. That is,
Hydrostatic pressure P1 at the sample introduction part
Isostatic pressure recovery unit hydrostatic pressure P2
Neutralization solution injection pressure P3
When
P1 = P3 (1)
P1> P2 (2)
Select to satisfy the following three formulas.
Heating by the heater may be performed so that all the channels have the same temperature. The heating result by the heater is the highest temperature at which boiling can be avoided, for example, 80 □. Since the sample and the buffer are always flowing, there is no formation of bubbles and no dry adhesion.
Next, the molecular weight separation unit 140 will be described. The number of molecular weight separation units 140 is provided according to the number of flow paths of the mixing unit 130. These have a plate-like structure with a width of about 1 mm. FIG. 7 is a diagram showing a schematic structure of one molecular weight separation unit. This molecular weight separation part is formed of a highly insulating plastic or quartz glass. Such a molecular weight separation portion can be formed by etching a substrate made of such a material, installing an electrode, and providing a lid. The migration region 141 is formed as a single flow path without partitioning in the height direction, while the collection unit 145 is composed of a plurality of flow paths partitioned in the height direction. The recovered component 150 is recovered from each recovery unit 145. Here, the recovery unit 145 adopts the same structure as that shown in FIG. 5 and has a sharply protruding cross-sectional shape or a curved surface protruding at a portion where each component is introduced. The buffer used for electrophoresis is indicated by 146 in the figure and is supplied from the entire side surface of the electrophoresis region 141. On the other hand, the sample is indicated by a sample introduction part 148 in the figure, and is introduced from the upper right corner of the migration region 141. In the sample introduced into the sample introduction part 148, the components are separated according to the molecular weight according to the electric field in the height direction.
Returning to FIG. 6, the supply method of the buffer and the mixed solution will be described. The right side view of FIG. 6 shows the arrangement pattern of the inlets of the mixed liquid channel 134 and the buffer channel 142. The mixed liquid channel 134 has one opening for each lane. On the other hand, the buffer flow path 142 has a linear inlet near the bottom surface of the support base 147. The mixed liquid channel 134 is supplied for each recovery lane and guided to the mixing unit 130. On the other hand, the buffer channel 142 communicates with the entire side surface of the molecular weight separation unit 140 and supplies a buffer (for example, 1 × TBE, pH 8.0) to the migration region 141.
FIG. 8 is a diagram for explaining how components are collected by the collection unit 145 of FIG. As shown, the recovered components are separated by two parameters: isoelectric point and molecular weight. The component for separation is collected in a specific flow path 152 according to its equipotential point and molecular weight.
FIG. 9 is a diagram illustrating a voltage application method in the molecular weight separation unit 140 and a state of separation of components corresponding thereto. The illustrated structure corresponds to the collection unit 145 of FIG. Here, it is important that a different voltage is applied to each recovery lane. If the ends of the recovery lanes are set to the same potential, the electric field of isoelectric focusing is disrupted and the separation efficiency is lowered. Here, the electrode on the cathode side of each recovery lane is selected along a potential gradient formed by isoelectric focusing. Next, as the potential on the anode side, a potential loaded with a voltage necessary for molecular weight electrophoresis is selected with respect to the potential on the cathode side. The voltage for molecular weight electrophoresis is preferably about 1000 to 3000 v for an electrophoresis lane of about 10 cm.
The collected components are guided to a predetermined container and collected by a tube or the like (not shown) connected to the collection unit 145.
Here, the recovered component may be guided to the sample concentrating device by a tube or the like (not shown) connected to the recovery unit 145. FIG. 14A is a diagram showing the main part of an apparatus that performs such sample concentration on a microchip. In this apparatus, after the sample is attached to the electrode, the applied sample is recovered by performing operations such as stopping the application of the electric field or reversing the potential. A potential opposite to the charged charge of the sample is applied to the electrode 334. The electrodes 334 are formed in the horizontal direction in the figure so as to be spaced apart from each other so that the sample can pass therethrough, and the sample flows from the gap. The interval D between the electrode plates 333 is arranged so as to be wider than the width W of the channel 112, more preferably twice or more the width of the channel 112. In this way, it is possible to prevent the phenomenon that the sample cannot enter between the electrode plates 333 due to the influence of the electric lines of force between the electrodes 334. Here, as shown in FIG. 14B, a configuration in which a counter electrode 335 is provided between a pair of electrodes 334 charged to a polarity opposite to the charge of the sample may be employed. Accordingly, the sample proceeds toward one of the electrodes 334 on both sides of the counter electrode 335, so that the amount of the sample attached to the electrode 334 can be increased.
After flowing the sample for a certain period of time in this way, an operation such as stopping the application of the electric field or inverting the electric potential is performed to collect the adhered sample. By doing so, it becomes possible to obtain the target component at a high concentration.
With the configuration described above, isoelectric point separation and molecular weight separation can be performed efficiently and continuously. The recovered component can be subjected to a protein identification sequence such as mass spectrometry.
Third Embodiment In the first and second embodiments, the separation apparatus that realizes the two-dimensional electrophoresis apparatus on the microchip has been described. That is, an example of a separation apparatus in which an isoelectric point separation unit and a molecular weight separation unit are integrated is shown. On the other hand, in the present embodiment, an example of a separation device including an isoelectric point separation unit and a data analysis unit is shown. FIG. 11 is a block diagram of the separation apparatus according to this embodiment. Light emitted from the light source 901 is applied to the electrophoresis lane of the isoelectric point separation device 903 via the spectroscope 902. Absorption or emission of the irradiated light is received by the light receiver 904. The received light is analyzed by data analysis 905, and component identification is performed by the component identification unit 906 with reference to information stored in advance.
Fourth Embodiment In the present embodiment, a separation apparatus that provides a sample for MALDI-TOFMS (Matrix-Assisted Laser Deposition Ionization-Time of Flight Mass Spectrometer). An example is shown. According to this apparatus, the sample can be fractionated for each molecular weight and used for MALDI-TOFMS analysis. Hereinafter, a procedure for preparing a sample for MALDI-TOFMS analysis will be described.
In order to perform MALDI-TOFMS measurement, it is necessary to reduce the molecular weight of the protein to be measured to about 1000 Da. For this reason, after reducing the molecular weight of the protein sample, it is mixed with a matrix solution, and then dried to obtain a measurement sample.
First, when the protein to be measured has an intramolecular disulfide bond, the reduction reaction is performed in a solvent such as acetonitrile containing a reducing reagent such as DTT (dithiothreitol). By doing so, the following decomposition reaction proceeds efficiently. In addition, after reduction, it is preferable to protect the thiol group by alkylation or the like and suppress oxidation again.
Next, a protein molecule that has been reduced using a protein hydrolase such as trypsin is subjected to molecular weight reduction treatment. Since the molecular weight reduction is performed in a buffer solution such as a phosphate buffer, desalting and removal of the polymer fraction, ie, trypsin, are performed after the reaction.
The obtained sample is mixed with a substrate of MALDI-TOFMS. Here, the matrix for MALDI-TOFMS is appropriately selected depending on the substance to be measured. For example, sinapinic acid, α-CHCA (α-cyano-4-hydroxycinnamic acid), 2,5-DHB (2 , 5-dihydroxybenzoic acid), a mixture of 2,5-DHB and DHBs (5-methoxysalicylic acid), HABA (2- (4-hydroxyphenylazo) benzoic acid), 3-HPA (3-hydroxypicolinic acid), Disranol, THAP (2,4,6-trihydroxyacetophenone), IAA (trans-3-indoleacrylic acid), picolinic acid, nicotinic acid and the like can be used.
Thereafter, concentration and drying are performed. The sample after drying is set in the MALDI-TOFMS apparatus together with the drying apparatus, applied with a voltage using an electrode provided in the drying apparatus, and irradiated with a nitrogen laser beam of, for example, 337 nm to perform MALDI-TOFMS analysis. FIG. 12 is a schematic diagram showing the configuration of the mass spectrometer. In FIG. 12, a dry sample is placed on a sample stage. The dried sample is irradiated with a nitrogen gas laser having a wavelength of 337 nm under vacuum. The dry sample then evaporates with the matrix. The sample stage serves as an electrode, and when a voltage is applied, the vaporized sample flies in a vacuum and is detected by a detection unit including a reflector detector, a reflector, and a linear detector.
FIG. 13A is a block diagram of a mass spectrometry system including the drying apparatus of the present embodiment. In this system, means 1002 for performing purification 1002 to remove impurities to some extent, separation 1003 to remove unnecessary components 1004, pretreatment 1005 of the separated sample, and drying 1006 of the sample after pretreatment are performed on the sample 1001. It has. Some or all of these means can be mounted on one or more microchips 1008. By continuously processing the sample on the microchip 1008, it is possible to efficiently and reliably identify a very small amount of component by a method with little loss.
As described above, in the processing flow of the present embodiment, the sample is continuously processed on one microchip 1008, whereby even a very small amount of components can be efficiently and reliably identified by a method with little loss. It becomes possible. Note that, as shown in FIG. 13B, all the pretreatment steps from sample purification 1002 to drying 1006 may be performed on one microchip 1008.
FIG. 10 shows a system configuration for realizing such a series of processing steps by one or more chips. This system includes a separation unit 810 including an isoelectric point separation unit 801 and a molecular weight separation unit 802, and a pretreatment unit 820 including a recovery unit 821, a reaction unit 822, a solvent replacement unit 823, a desalting unit 824, and a drying unit 825. Become. Only the separation unit 810 may be realized on one chip, or the separation unit 810 and the preprocessing unit 820 may be realized on one chip. By using such a system, a sample suitable for mass spectrometry including MALDI-TOFMS can be provided.
The present invention has been described based on the embodiments. It should be understood by those skilled in the art that these embodiments are exemplifications, and that various modifications can be made to the combination of each component and each manufacturing process, and such modifications are also within the scope of the present invention.

Claims (11)

A separation apparatus comprising an isoelectric point separation region in which a pH gradient is formed, a pair of electrodes for applying an electric field to the isoelectric point separation region, and a sample introduction unit for introducing a sample into the isoelectric point separation region. And
A separation apparatus, wherein the sample is introduced from the side in the electric field application direction through the sample introduction unit. 2. The separation apparatus according to claim 1, wherein the sample introduction unit includes: a flow channel into which a sample is introduced; a partition wall interposed between the flow channel and the isoelectric point separation region;
And a plurality of introduction flow paths communicating with the flow path and the isoelectric point separation region are formed in the partition wall. The separation device according to claim 1 or 2, wherein the isoelectric point separation region has a rectangular shape, the pair of electrodes are provided along a pair of opposing sides of the rectangular shape, and one of the remaining sides A separation apparatus characterized in that the sample introduction section is provided along the side of the. The separation apparatus according to any one of claims 1 to 3, further comprising a sample collection unit that communicates with the isoelectric point separation region and collects a sample introduced into the isoelectric point separation region. apparatus. 5. The separation apparatus according to claim 4, wherein the sample recovery unit is disposed to face the sample introduction unit. 6. The separation apparatus according to claim 1, wherein the sample collection unit includes a plurality of collection lanes communicating with the isoelectric point separation region. The separation device according to claim 6, wherein the collection ports of the plurality of collection lanes are densely arranged in a line along the isoelectric point separation region. 8. The separation device according to claim 6 or 7, wherein the wall section defining the plurality of recovery lanes protrudes sharply toward the isoelectric point separation region in a plane in which the isoelectric point separation region extends. A separation device having a cross-sectional shape or a shape in which a curved surface protrudes. The separation device according to any one of claims 1 to 8, wherein a light irradiation unit that irradiates light to a predetermined portion of the isoelectric point separation region, and a light detection unit that detects light absorption or light emission at the light irradiation portion. And an analysis unit that analyzes components contained in the sample based on the light absorption or emission. The separation apparatus according to any one of claims 1 to 9, further comprising a molecular weight separation unit that performs component separation based on molecular weight adjacent to the isoelectric point separation region, wherein isoelectric point separation and molecular weight separation are performed continuously. A separation apparatus configured to be executed on Separation means for separating a biological sample according to molecular size or property, pretreatment means for performing pretreatment including enzymatic digestion on the sample separated by the separation means, and drying for drying the pretreated sample Means, mass spectrometry means for mass spectrometry of the dried sample,
And the separation means includes the separation device according to any one of claims 1 to 10.

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