JPWO2005111572A1 - Sample preparation apparatus and method - Google Patents

Abstract

An object of this invention is to provide the sample preparation apparatus etc. which can respond | correspond to a various protocol, can improve reproducibility, and can obtain a reliable analysis result. A sample preparation device according to the present invention prepares a specimen sample from a substance containing a biomolecule, and contains a first container in which the substance is stored and a solution to be injected into the first container. A second container, a dispensing means for injecting the solution contained in the second container into the first container, a movement holding means for holding the first container movably, and a movement holding means. A shaking means for stirring the substance and solution in the first container, and a collecting means for discharging the shaken solution from the first container and collecting the specimen sample third.

Description

  The present invention relates to an apparatus and method for preparing an analyte sample from a substance such as a carrier containing a biomolecule.

  Proteome analysis is attracting attention as an important research in the post-genomic era. In proteome analysis, comprehensive analysis of proteins contained in cells constituting the living body is performed, and in recent years, protein profiling using mass spectrometry and protein separation by gel electrophoresis represented by two-dimensional electrophoresis has been frequently performed. Has been done. As a typical analysis procedure, first, a biological sample prepared for electrophoresis is separated on a polyacrylamide gel, stained and image-analyzed, and a spot of each protein is cut out based on the result. Next, after subjecting the cut-out spot to in-gel protein enzyme digestion, a sample containing a digested peptide fragment as an analyte sample for mass spectrometry is collected using a solution reagent or the like. Then, mass analysis of the peptide fragment was performed, and the theoretical mass spectrum of the peptide fragment predicted from the database was compared with the pattern of the actually measured mass spectrum using a protein identification search engine. To identify proteins.

  Here, the analysis sensitivity, the accuracy of identification, and the reproducibility when finally performing protein identification by mass spectrometry are a series of steps from preparation of a sample sample for mass spectrometry from a spot separated by electrophoresis. The place depends on the processing of. In addition, various automatic instruments for sample preparation have been developed from the viewpoints of processing a large number of specimens to improve the throughput of analysis and reliably increasing reproducibility.

  For example, Non-Patent Document 1 discloses a gel enzyme digestion processing apparatus that automatically digests a gel plug containing a protein recovered from a two-dimensional electrophoresis gel in a plate and then dispenses the peptide to another plate. (Product name: Ettan Digester) is described. Non-Patent Document 2 describes a laboratory automation system (product name: BIOMEK (registered trademark) 2000) in which a series of operations such as sampling, dispensing, dilution, and plate washing are automated.

Furthermore, Non-Patent Document 3 discloses a mass spectrometry pretreatment automation device (product name: MultiPROBE II) that automates in-gel enzyme digestion, desalting / concentration, and spotting on a mass spectrometer target plate of a protein isolated by electrophoresis. ) Is described. Furthermore, Non-Patent Document 4 includes an in-gel protein digestion automation system (product name: Investigator ProGest) that automatically digests proteins separated by electrophoresis in a gel, and a sample preparation device for mass spectrometry (product name: Investigator ProMS). ), Sample preparation automation system (product name: Investigator ProPrep) and the like.
Amersham Biosciences, Inc. “Spot Picking & Enzyme Treatment Gel Enzyme Digestion Equipment” Brochure Beckman Coulter Co., Ltd. BIOMEK (registered trademark) 2000 Laboratory Automation System PerkinElmer Japan Co., Ltd. “Life Science Budget Application Catalog 2003 → 2004” Pamphlet, page 26, lower column (Mass Spectro Pretreatment Automation System MultiPROBE II) NIPPON Technolactas Co., Ltd. “Investigator ProGest (trademark) Protein Digestion Station”

  However, with various conventional automated devices, the recovery rate of the sample for mass spectrometry is inadequate. In particular, depending on the properties of the sample, the required sensitivity, and the type of protocol, the preparation of the sample for mass spectrometry is difficult for skilled workers. There were many things that I had to rely on. In this case, the recovery rate of the sample for mass spectrometry may vary depending on the worker and the working situation. As a result, in mass spectrometry, variations in analysis results based on such differences (decrease in identification accuracy) and reproducibility have been problems. In particular, recently, for example, the need for highly sensitive microanalysis for identifying proteins with low expression levels has further increased, and the problems of variation in analysis results and reproducibility tend to become more apparent.

  Therefore, the present invention has been made in view of such circumstances, can cope with various protocols, sufficiently suppress such variations, improve reproducibility, and obtain highly reliable analysis results. It is an object of the present invention to provide a sample preparation apparatus and method capable of performing the above.

  In order to solve the above problems, a sample preparation apparatus according to the present invention is a substance containing a biomolecule (for example, a sample in which a biomolecule is supported on a carrier such as a gel spot of various proteins separated and extracted by electrophoresis, and the like. A liquid or solid sample or the like; hereinafter referred to as a “biomolecule-containing substance”)) for preparing a specimen sample, and a first container for containing the biomolecule-containing substance; A second container for storing a solution to be injected into the first container; a dispensing means for injecting the solution stored in the second container into the first container; and the first container is movable. Moving holding means for holding, shaking means for shaking the holding means to stir the biomolecule-containing substance and solution in the first container, and discharging the shaken solution from the first container Collect the specimen sample in a third container And a yield means.

  In the sample preparation device configured as described above, the biomolecule-containing substance is accommodated in the first container. On the other hand, the second container contains an appropriate solution to be injected into the first container. This solution is injected into the first container from the second solution by means of dispensing. The first container is moved to a desired position by the movement holding means and is held in that state.

  The first container moved and held at an appropriate position is shaken by the shaking means, whereby the substance and the solution containing the biomolecule in the first container are agitated. At this time, you may make it shake a 1st container via a movement holding means. In this way, the biomolecule contained in the biomolecule-containing substance and the solution are sufficiently mixed. Therefore, compared to simply injecting the solution into the biomolecule-containing substance and performing a treatment such as incubation in a stationary state, the contact frequency between the biomolecule in the biomolecule-containing substance and the solution is increased, and the chemical reaction or The chemical operation is prevented from proceeding unevenly. Furthermore, various salts that hinder mass spectrometry can be efficiently washed away. In addition, since the first container is moved and a desired shaking operation is performed at a desired position, and other operations are possible if necessary, a plurality of treatments, that is, a plurality of types of solutions and a biomolecule-containing substance A series of processes in a protocol in which the interactions are sequentially performed can be automatically processed continuously.

  Further, the solution after the shaking treatment is discharged from the first container, and the solution interacting with the biomolecule in the biomolecule-containing substance is used as a specimen sample for another processing operation. Collected in three containers. Such a recovery process can also be performed by moving the first container to an appropriate desired location by the moving holding means, for example, so that it becomes easy to equip a mechanism, device, etc. for recovery.

  Moreover, it is preferable that a 2nd container accommodates the several solution from which a kind mutually differs separately. In the normal protocol procedure, various solutions (for example, chemical reagents, digestive enzymes, etc.) are supplied to the biomolecule-containing substance for each step and incubated under predetermined conditions, and then the solutions are collected each time. If various solutions necessary for such a protocol are accommodated in the second container, the interaction between each solution and the biomolecule-containing substance is preferably carried out using the movement holding means and the shaking means. .

  Furthermore, it is more preferable that the shaking means is one in which the stirring speed is adjusted according to the type of the solution. The mixing conditions for each solution and the biomolecule-containing substance vary depending on their interaction. Therefore, the sample preparation apparatus according to the present invention has a sufficient interaction between the biomolecule in the biomolecule-containing substance and the solution by adjusting the stirring speed of the shaking means to a constant value according to each solution. And it is implemented with good reproducibility.

  More specifically, an area where the first container is placed and an area where the second container is placed are connected in series, and an area where the shaking is performed and an area where the specimen sample is collected However, it is more preferable that the region where the first container is placed and the region where the second container is placed are provided side by side. In this way, it is possible to separate the area (area) where the dispensing operation from the second container to the first container, which is a so-called static process, and the area where the shaking operation, etc., which is a dynamic process, are performed. Therefore, it is preferable in process management.

  In addition, it is more preferable to further include a pressurizing means for supplying gas into the first container containing the solution after being shaken to pressurize the inside of the first container. The pressurizing means preferably includes a sealing means (for example, an elastic body such as silicon rubber) at the time of pressurization in order to pressurize more efficiently.

  Further, the sample preparation method according to the present invention is a method that is effectively carried out using the sample preparation device according to the present invention, and is a method for preparing a specimen sample from a biomolecule-containing substance, the biomolecule containing An accommodating step for accommodating the substance in the first container, a solution accommodating step for accommodating the solution injected into the first container in the second container, and the solution accommodated in the second container in the first container A dispensing step of injecting into the first container, a moving and holding step of moving and holding the first container to a predetermined region, and a shaking for stirring the holding means and stirring the biomolecule-containing substance and solution in the first container. After performing the shaking step and the shaking step, the method includes a recovery step of discharging the solution from the first container and recovering the specimen sample in the third container.

  In this case, in the solution storage step, it is useful to use a second container in which a plurality of different types of solutions are individually stored.

  Furthermore, in the recovery step, it is preferable to supply gas to the first container and pressurize the inside of the first container. In the recovery step, it is preferable to use a sealing means (for example, an elastic body such as silicon rubber) during pressurization.

  According to the sample preparation apparatus and method of the present invention, the biomolecule-containing substance contained in the first container is supplied with the solution from the second container, and then the first container is moved and held by the moving holding means. Further, the first container is sufficiently agitated by shaking under the desired conditions. And since the solution is collect | recovered as a specimen sample, extraction separation of the target component from a biomolecule containing substance is implemented with high efficiency, and the reproducibility of processing operation is extremely improved. Therefore, processing corresponding to various protocols can be realized, variation in the analytical processing in which the specimen sample is provided is sufficiently suppressed, and the reproducibility is improved to obtain a highly reliable analysis result. Is possible.

  Hereinafter, embodiments of the present invention will be described in detail. The positional relationship such as up, down, left, and right is based on the positional relationship shown in the drawings unless otherwise specified. Further, the dimensional ratios in the drawings are not limited to the illustrated ratios.

  FIG. 1 is a front view schematically showing a preferred embodiment of a sample preparation apparatus according to the present invention. 2 is a cross-sectional plan view taken along line II-II in FIG. FIG. 3 is a right side view of the sample preparation device shown in FIG. The processing apparatus 100 (sample preparation apparatus) is a two-dimensional electric device that uses a pretreatment apparatus for preparing a specimen sample to be subjected to mass spectrometry, for example, a peptide mass fingerprinting (PMF) method using in-gel enzyme digestion described later. It is the pre-processing apparatus for preparing the specimen sample used for the mass spectrometry for protein spot identification isolate | separated by electrophoresis. In the processing apparatus 100, a chip disposal unit 3, a chip stage 4, reagent reservoirs 5 and 6 (both are second containers) are arranged in a row on a base 2 in a housing 1, and the chip temperature The adjusting unit 7 and the vacuum manifolds 8 and 9 are provided together.

  The chip stage 4 is for mounting a chip holder 41 on which a plurality of chips 42 are arranged, for example, in an array. The chip 42 has an elongated and substantially cylindrical shape, and the number of chips 42 arranged on the chip holder 41 is not particularly limited. However, in the illustrated example, chips having a capacity of 200 μL are arranged in 8 columns × 12 rows (the depth of the processing apparatus 100). The direction orthogonal to the direction is the 'row' direction. In addition, the reagent reservoir 5 (second container) has a recess 51 that is wide and narrow in the horizontal cross section, and an appropriate reagent (solution) is accommodated in the recess 51. It has become. In addition, the reagent reservoir 5 functions as a thermostat that holds the reagent stored in the recess 51 at a constant temperature, for example, room temperature.

  Further, the reagent reservoir 6 (second container) is provided with a plurality of (in the illustrated example, 10) recesses 61 having the same shape as the recesses 51 in the longitudinal direction of the processing apparatus 100. Appropriate reagent solutions (solutions) different from each other are accommodated. Furthermore, the reagent reservoir 6 functions as a thermostat that holds the reagents contained in the recesses 61,... At a low temperature such as 4 ° C. (4 ° C. incubation).

  A dispensing head 10 (dispensing means) is provided vertically above the chip discarding unit 3, the chip stage 4, and the reagent reservoirs 5 and 6 arranged in a row. The dispensing head 10 includes a dispensing syringe probe 11 arranged in parallel in the depth direction by the same number as the tips 42,... (Eight in the illustrated example). The dispensing head 10 itself is driven in the row direction (arrow X direction in the figure) and the depth direction (arrow Y direction in the figure), and the syringe probe 11 is driven in the vertical direction (arrow Z direction in the figure). It is like that. The driving method of the dispensing head 10 is not particularly limited, and for example, a one-way stage or an XY stage having a known guide rail provided above the dispensing head 10 can be used. Also, the driving method of the syringe probe 11 in the vertical direction is not particularly limited, and a known motor device can be used, or an XYZ stage movable with the dispensing head 10 may be used.

  Further, a grip shaker unit 15 is provided adjacent to the chip temperature control unit 7 and the vacuum manifolds 8 and 9 at the rear of the apparatus. The gripping and shaking unit 15 can hold a filter plate 81 (holding tool) on a stage 151 driven in a row direction (X direction in the drawing) and is an arm-shaped robot driven in the depth direction (Y direction in the drawing). A hand 152 (holding means) is attached. In addition, the gripping and shaking unit 15 includes a PZT element 153 that contacts the robot hand 152. As a result, the filter plate 81 held by the robot hand 152 is shaken at a predetermined shaking speed. As described above, the gripping and shaking unit 15 serves as both the movement holding means and the shaking means of the present invention.

  Further, the gripping and shaking unit 15 is provided with a cover (not shown) that can be opened and closed and covers the entire upper part of the filter plate 81 gripped by the robot hand 152. With the cover closed, the filter plate 81 is sealed and shielded from light, and the sealed space is sealed by nitrogen gas supplied from a nitrogen gas source (not shown) connected to the nitrogen gas supply port 21. Is purged with nitrogen.

  Here, the filter plate 81 includes, for example, a plate in which a plurality of wells 82 (first containers) are arranged. The number of arrangement of the plurality of wells 82 is not particularly limited, and for example, 96 are arranged in 8 columns × 12 rows. The well 82 preferably has a filter 83 on the bottom surface. Examples of the filter 83 include a PVDF membrane.

  On the other hand, the chip temperature adjusting unit 7 is for placing the filter plate 81 moved by the gripping and shaking unit 15 and the wells 82,. The contents of the (container) function as a thermostatic chamber that maintains a temperature such as 37 ° C. (37 ° C. incubation). Further, the vacuum manifold 9 is connected to an exhaust port 20 to which a vacuum exhaust system (not shown) is connected, and the contents of each well 82 on the filter plate 81 moved by the grip shaker unit 15 are so-called “ It is for recovering from the well 82 by “evacuation”. Further, the vacuum manifold 8 is also connected to an exhaust port 20 connected to a vacuum exhaust system (not shown), and discharges residual liquid in the well 82 and performs vacuum cleaning.

  Further, the housing 1 is provided with a front door 1a in which a rod-like handle portion H is installed. In a state where the front door 1a is pushed upward, the space above the base 2 is opened, and the front door 1a is opened. The space above the base 2 is blocked from the outside while being pushed downward. Further, a glass window W1 is provided on the front door 1a, and further, a glass window W2 is provided on the side wall of the housing 1, thereby enabling the inside to be visually recognized.

  Further, the front panel 30 below the front door 1a is inclined so as to protrude downward, and is provided with an emergency stop button 31, a touch panel 32, a decompression monitor 33 in the vacuum manifolds 8 and 9, and the like. Further, a side surface 34 communicating with the front panel 30 is provided with a nitrogen gas pressure gauge 35, a heat exhaust port 36, a temperature regulator 37 of the chip temperature regulator 7, and a temperature regulator 38 of the reagent reservoir 6. .

  An example of a procedure for carrying out an embodiment of the sample preparation method according to the present invention using the processing apparatus 100 configured as described above will be described below. This procedure is a method of preparing an analyte sample for mass spectrometry by extracting peptides from protein spots separated by two-dimensional electrophoresis by the PMF method using in-gel enzyme digestion.

  First, as a biomolecule-containing substance, a protein spot gel piece is cut out and washed. Specifically, a plurality of protein spots that are not unevenly distributed in isoelectric point or molecular weight and have a relatively high content are selected from a polyacrylamide gel subjected to two-dimensional electrophoresis by image processing or the like. Next, the gel piece containing the selected protein spot is cut out.

  Next, each gel piece (an example of a biomolecule-containing substance) is accommodated in each well 82 of the filter plate 81 (accommodating step). The filter plate 81 is placed on the vacuum manifold 8. In parallel with this, the following reagents are prepared and stored in the recesses 51, 61,... At appropriate positions of the reagent reservoirs 5, 6 (solution storage step; hereinafter stored in the reagent reservoir 6). To explain.)

・ Tris Buffer solution adjusted to pH 8.5
・ Reducing liquid ・ Alkylating liquid ・ Water / methanol / acetic acid = 4: 5: 1 mixed liquid ・ Ammonium bicarbonate ・ Destained liquid ・ Acetonitrile 100% liquid ・ Trypsin (0.1% octyl glycoside, or 0. 1% 5-cyclohexyl-pentyl-beta-D-maltoside)
A mixture of acetonitrile / water / 0.1% trifluoroacetic acid (TFA) = 1: 1 A mixture of acetonitrile / water / 0.1% trifluoroacetic acid (TFA) = 7: 3

  Next, destaining is performed prior to in-gel digestion. Specifically, first, the dispensing head 10 is moved so that the syringe probes 11,... Of the dispensing head 10 are positioned on the tip 42 placed on the tip holder 41. Further, the syringe probes 11 are moved downward, and the tips 42 are attached to the syringe probes 11. Next, the dispensing head 10 is moved from the initial position so that the syringe probes 11,... Of the dispensing head 10 are located in the destaining liquid stored in the reagent reservoir 6. Next, the plurality of syringe probes 11,... Are moved downward until the tips of the tips 42 are immersed in the destaining solution, stopped, and after sucking an appropriate amount (for example, 200 μL), the syringe probes 11,. Move upward to reach the initial position in the direction.

  Then, the dispensing head 10 is moved so that the syringe probes 11,... Of the dispensing head 10 are positioned on the respective wells 82 of the filter plate 81 placed on the vacuum manifold 8. Further, the syringe probes 11,... Are moved downward, and the destaining solution that has been sucked and held inside the tip 42 is injected into the well 82 (dispensing step). This dispensing operation is the same even if the solutions are different, and will not be described in detail in the following description.

  This dispensing operation is repeated, and the destaining solution is dispensed into all the wells 82 on the filter plate 81. Next, the filter plate 81 is gripped by the gripping and shaking unit 15, the stage 151 and the robot hand 152 are driven, and the filter plate 81 is placed at a predetermined position of the chip temperature adjusting unit 7 (moving and holding step). In this gripping state, the PZT element 153 is operated, and the gel pieces and destaining solution stored in the well 82 are stirred for 5 minutes at a relatively high shaking speed (shaking process). Hereinafter, shaking stirring under a relatively low speed condition is referred to as “low speed stirring”, and shaking stirring under a relatively high speed condition is referred to as “high speed stirring”.

  After shaking, the gripping shaking unit 15 is driven, the filter plate 81 is transferred to the vacuum manifold 8, and the vacuum exhaust system is operated to suck and discharge the destaining solution remaining in the well 82. Then, the gripping and shaking unit 15 is driven, and the filter plate 81 is transferred to the chip temperature adjusting unit 7 or the vacuum manifold 8.

  Since the operations up to the above dispensing, shaking and agitation and aspiration and discharge are similarly performed on other reagent solutions, the overlapping contents are appropriately omitted in the following description.

  Next, a reducing solution is dispensed instead of the destaining solution, and after stirring at room temperature for 30 minutes, the remaining solution is sucked and discharged. Subsequently, the alkylating solution is dispensed, stirred at high speed for 30 minutes at room temperature, and then the remaining solution is sucked and discharged. By these treatments, the proteins contained in the gel pieces are reduced and alkylated.

  Then, a water / methanol / acetic acid mixture is dispensed, stirred at high speed for 30 minutes, and a series of treatments of sucking and discharging the remaining liquid is repeated 4 to 5 times, and then ammonium bicarbonate is dispensed for 3 minutes. After stirring at low speed, the gel pieces are equilibrated by sucking and discharging the remaining liquid.

  Next, the gel pieces that have been reduced, alkylated, washed and equilibrated are subjected to in-gel digestion. Specifically, acetonitrile is dispensed and stirred at high speed for 5 minutes, and then the remaining liquid is sucked and discharged to dehydrate the gel pieces. Then, trypsin is dispensed, for example, about 0.5 to 3 μL, and allowed to stand for 5 to 10 minutes to swell the gel piece.

  Next, about 10 to 20 μL of ammonium bicarbonate is dispensed, for example, and kept at 37 ° C. for about 5 to 12 hours, and then a mixture of acetonitrile / 0.1% TFA aqueous solution = 1: 1 is dispensed, for example, 100 μL. After stirring for 15 minutes at high speed, the gripping and shaking unit 15 is driven, the filter plate 81 is transferred to the vacuum manifold 9, and the vacuum exhaust system is operated to collect the solution (analyte sample) in the well 82. Aspirate and collect in the upper well 92 (third container) (collection process). Further, a mixed solution of acetonitrile / 0.1% TFA aqueous solution = 7: 3 was stirred at high speed for 15 minutes, and then the solution in the well 82 (analyte sample) was removed from the well 92 on the recovery plate 91 by the vacuum manifold 9. Collect by suction (collection process). As described above, in-gel digestion and the resulting peptide are extracted into the recovered solution.

  Then, these collected liquids collected from the processing apparatus 100 as the specimen sample are combined and centrifugally concentrated to about 1 to 5 μL, for example. Furthermore, after completely drying it, it melt | dissolves in the 5% acetonitrile / 0.1% TFA liquid mixture (for example, about 5 microliters) prepared separately, and obtains the sample for mass spectrometry. Note that the chip 42 that is no longer needed is discarded to the chip discarding unit 3.

  According to the processing apparatus 100 configured as described above and the sample preparation method of the present invention using the processing apparatus 100, a reagent solution corresponding to the protocol is placed on the gel pieces accommodated in the wells 82,. The sample is dispensed and transferred to the chip temperature adjusting unit 7 together with the filter plate 81 by the gripping and shaking unit 15. Then, shaking and stirring are performed at an appropriate stirring speed. Thereby, the protein molecules contained in the gel piece and the reagent solution are sufficiently mixed in a series of automated processes.

  Therefore, compared with simply injecting the reagent solution into the gel piece and performing a treatment such as incubation in a stationary state, the contact frequency between the protein molecule in the gel piece and the solution is increased, and the chemical reaction and chemical operation are performed. Uneven progress can be suppressed, and operations such as desalting and washing can be performed efficiently. Therefore, the concentration of the target component in the specimen sample finally extracted from the gel piece can be increased and the reproducibility of the extraction efficiency can be improved. Thereby, even in mass spectrometry performed using a specimen sample, an analysis result having high sensitivity, high accuracy, high accuracy, and high reproducibility can be obtained. Moreover, since it is based on an automation process, it is possible to suppress a decrease in reproducibility caused by a difference in operator skills.

  Further, the filter plate 81 is shaken in a state where the filter plate 81 is held at the tip of the arm-like robot hand 152 of the gripping and shaking unit 15, that is, in a state where the distance is larger than the base of the gripping and shaking unit 15. The contents in the filter plate 81 can be easily stirred more vigorously. Therefore, the protein molecules contained in the gel piece and the reagent solution are mixed better, and the extraction efficiency can be further improved.

  Furthermore, since the filter plate 81 is appropriately transferred to the vacuum manifolds 8 and 9 by the grasping and shaking unit 15, the residual liquid in the well 82 can be aspirated and discharged and the specimen sample can be aspirated and collected on the spot. It is possible to increase the layout design margin of a mechanism for collecting / discharging and cleaning the solutions such as the vacuum manifolds 8 and 9. Furthermore, since a plurality of different types of reagent solutions can be stored in the reagent reservoir 6, it is possible to improve the processing performance (throughput) when performing a protocol that requires various reagent solutions for each step. In addition, since the agitation speed can be adjusted with high reproducibility according to the type of reagent solution by the gripping and shaking unit 15, the recovery efficiency (chemical yield) of the final recovery sample and the reproducibility of the recovery amount can be further improved. it can.

  In addition, a chip disposal unit 3, a chip stage 4, and reagent reservoirs 5 and 6 (both are second containers) are arranged in a row on the base 2 in the housing 1, and the chip temperature control unit 7. Since the vacuum manifolds 8 and 9 are provided side by side, a region (area) where the filter plate 81 is left stationary, that is, a dispensing operation that is a static process, and a filter plate 81 is shaken. This is preferable in terms of process management because it can be separated from an operation area that is a dynamic process. In addition, by performing nitrogen gas purging in the chip temperature control unit 7, since a shaking operation can be executed in an inert atmosphere, it is possible to suppress inconvenient reactions such as oxidation by oxygen in the air, The reproducibility of the concentration of the extracted component in the collected sample can be more reliably maintained.

  Further, since the filter plate 81 is gripped by the robot hand 152 from the side (lateral direction), the vacuum manifolds 8 and 9 can be positioned directly below the filter plate 81, thereby supporting the filter plate 81 from below. As compared with the case, the reagent solution in the well 82 can be easily collected.

  Here, FIG. 4 is a right side cross-sectional view schematically showing a main part of another preferred embodiment of the sample preparation device according to the present invention. The processing apparatus 200 (sample preparation apparatus) is configured in the same manner as the processing apparatus 100 except that it includes a nitrogen gas pressurizing head 70 (pressurizing means).

  The nitrogen gas pressurizing head 70 includes nozzles 71 arranged in parallel in the depth direction (the arrow Y direction in the figure) as many as the wells 82 (... Are omitted in the example shown in the figure). The nitrogen gas pressurizing head 70 itself is driven in the column direction (arrow X direction in FIG. 2) and the depth direction (arrow Y direction in the figure), and the nozzle 71 in the vertical direction (arrow Z direction in the figure). It is designed to be driven. The driving method of the nitrogen gas pressurizing head 70 is not particularly limited as in the case of the dispensing head 10. For example, a one-way stage or an XY stage having a known guide rail provided above the nitrogen gas pressurizing head 70. Etc. can be used. Also, the driving method of the nozzle 71 in the vertical direction is not particularly limited, and a known motor device can be used, or an XYZ stage movable with the nitrogen gas pressurizing head 70 may be used.

  An annular groove R is formed at the tip (lower end in the figure) of the nozzle 71, and an annular ring 72 (sealing means) formed of an elastic body such as silicon rubber is fitted into the annular groove R. Are combined. Further, the nozzle 71 has a taper formed at the tip of the annular ring 72, and the tip is inserted into a well 82 juxtaposed on the filter plate 81. Furthermore, a nitrogen gas supply pipe 73 connected to the nitrogen gas supply port 21 (see FIG. 2) is connected to the other end (illustrated upper end) of the nozzle 71 via a joint 74.

  According to the processing apparatus 200 configured as described above, the filter plate 81 including the well 82 in which the gel piece G and the reagent solution L are accommodated and shaken is gripped by the robot hand 152 and the vacuum manifold 9 is placed on the vacuum manifold 9. Then, the nitrogen gas pressurizing head 70 is moved to a predetermined position above the vacuum manifold 9 so that the nozzles 71 of the nitrogen gas pressurizing head 70 are positioned on each well 82. Then, the nozzles 71 are moved downward, the tip of the nozzle 71 is inserted into the well 82, the annular ring 72 is brought into contact with the upper edge of the well 82, and the upper open end of the well 82 is sealed with the annular ring 72. Stop.

  At this time, the tip of the nozzle 71 including the annular ring 72 is urged in advance by an elastic body such as a spring, and the annular ring 72 moves toward the well 82 while the annular ring 72 is in contact with the upper edge of the well 82. It is preferable that the pressing is performed with a predetermined pressing pressure because the adhesion between the well 82 and the annular ring 72 and the sealing inside the well 82 are further enhanced.

In this state, nitrogen gas (N ₂ ) is supplied to each nozzle 71 at a predetermined pressure through a nitrogen gas supply pipe 73 from a nitrogen gas source (not shown). Nitrogen gas Ng flowing through the internal space K of the nozzle 71 is sent into the well 82 from the tip of the nozzle 71. Thereby, the inside of the well 82 is pressurized by the nitrogen gas Ng, and the sample solution L is pushed downward from the lower open end 82 a of the well 82. Then, the sample solution L passes through the filter 83 and is recovered in the well 92 of the recovery plate 91 in the vacuum manifold 9 (recovery step).

  At this time, since the upper end of the well 82 is sealed by the annular ring 72 and the inside of the well 82 is hermetically sealed, the pressure of the supplied nitrogen gas Ng is not dissipated to the outside of the well 82 and the internal pressure of the well 82 is Is sufficiently enhanced. Therefore, the recovery efficiency of the reagent solution L stored in the well 82 can be further increased. Therefore, the processing apparatus 200 including the nitrogen gas pressurizing head 70 is particularly useful for proteomic analysis (proteomics) in which the recovery efficiency of the sample is particularly important.

  Note that the processing apparatus 200 also has the same operational effects as the processing apparatus 100 described above, but detailed description thereof is omitted here to avoid redundant description.

  The present invention is not limited to the above-described embodiment, and various modifications can be made without changing the gist thereof. For example, the sample processed by the processing apparatuses 100 and 200 is not limited to a gel piece, but may be a solid (body) or liquid (body) sample other than the gel piece. Moreover, the processing apparatuses 100 and 200 are not limited to processing operations involving in-gel digestion, and can be applied to various pretreatments of other carriers including biological samples. Furthermore, the number of the wells 82 held by the filter plate 81 and the wells 92 of the recovery plate 91, the number of the concave portions 61 provided in the reagent reservoir 6, the layout on the base 2, and the like are not limited to those illustrated. Furthermore, the syringe 11 and the nozzle 71 may be shared and provided integrally, or may be provided separately as separate bodies.

  Moreover, the kind of various reagent solution used for the protocol demonstrated in embodiment, those specific compositions, mixing ratios, and the usage-amount are an illustration to the last, Comprising: This invention is not limited to them. Furthermore, 0.1% octyl glycoside or 0.1% 5-cyclohexyl-pentyl-beta-D-maltoside was listed as an example of the surfactant used for trypsin in the reagent solution. Among these, 0.1% 5-cyclohexyl-pentyl-beta-D-maltoside is more preferable.

  As described above, the sample preparation apparatus and method according to the present invention includes a moving holding means for holding the first container movably, a biomolecule-containing substance in the first container by shaking the moving holding means, and Since it is equipped with a shaking means for stirring the solution, it can support various protocols, sufficiently suppress variations that have been a problem in conventional sample pretreatment, and improve reproducibility and reliability. High analysis results can be obtained. Therefore, it can be widely used for analysis of substances containing biomolecules and sample preparation therefor.

1 is a front view schematically showing a preferred embodiment of a sample preparation device according to the present invention. FIG. 2 is a cross-sectional plan view taken along line II-II in FIG. 1. It is a right view of the sample preparation apparatus shown in FIG. It is right side sectional drawing which shows roughly the principal part of other suitable embodiment of the sample preparation apparatus by this invention.

Claims (12)

An apparatus for preparing an analyte sample from a substance containing a biomolecule,
A first container containing the substance;
A second container containing a solution to be injected into the first container;
Dispensing means for injecting the solution contained in the second container into the first container;
Moving holding means for holding the first container movably;
Shaking means for stirring the substance and the solution in the first container by shaking the moving holding means;
Recovery means for discharging the solution after the shaking from the first container and recovering the specimen sample in a third container;
A sample preparation device comprising: The second container is for individually storing a plurality of different types of solutions.
The sample preparation device according to claim 1. In the shaking means, the stirring speed is adjusted according to the type of the solution.
The sample preparation apparatus according to claim 2. An area where the first container is placed and an area where the second container is placed are connected,
The region where the shaking is performed and the region where the specimen sample is collected are provided side by side with the region where the first container is placed and the region where the second container is placed,
The sample preparation device according to any one of claims 1 to 3. Pressurization means for supplying gas into the first container containing the solution after being shaken to pressurize the first container;
The sample preparation device according to any one of claims 1 to 4. The substance containing a biomolecule is a carrier containing a biomolecule,
The sample preparation device according to any one of claims 1 to 5. The shaking means has arm-shaped holding means.
The sample preparation device according to any one of claims 1 to 6. The shaking means holds the first container or the holder of the first container from the side.
The sample preparation device according to any one of claims 1 to 7. A method for preparing an analyte sample from a substance containing a biomolecule,
A carrier containing step of containing the substance in a first container;
A solution storage step of storing a solution to be injected into the first container into a second container;
A dispensing step of injecting the solution contained in the second container into the first container;
A moving and holding step of moving and holding the first container to a predetermined region;
A shaking step of shaking the holding means to stir the substance and the solution in the first container;
A recovery step of discharging the solution from the first container and recovering the analyte sample in a third container after performing the shaking step;
A sample preparation method comprising: In the solution storage step, as the second container, a container in which a plurality of different types of solutions are individually stored,
The sample preparation method according to claim 9. In the recovery step, gas is supplied to the first container to pressurize the first container.
The sample preparation method according to claim 9 or 10. Using a carrier containing a biomolecule as the substance containing the biomolecule,
The sample preparation method according to any one of claims 9 to 11.

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