CN101156064A - Method of automatic sample processing for microchip with sealing lid for bioanalysis and apparatus for automatic sample processing - Google Patents

Method of automatic sample processing for microchip with sealing lid for bioanalysis and apparatus for automatic sample processing Download PDF

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Publication number
CN101156064A
CN101156064A CNA2006800116444A CN200680011644A CN101156064A CN 101156064 A CN101156064 A CN 101156064A CN A2006800116444 A CNA2006800116444 A CN A2006800116444A CN 200680011644 A CN200680011644 A CN 200680011644A CN 101156064 A CN101156064 A CN 101156064A
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China
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baseplate part
lid sealing
sealing
microchip
passage
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CNA2006800116444A
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Chinese (zh)
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藤田真知子
川浦久雄
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NEC Corp
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NEC Corp
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Abstract

This invention provides a method for automating a procedure comprising carrying out electrophoretic separation of a sample liquid as an analyte and then separating and removing a lid part bonded and fixed on the upper surface of a substrate part constituting a lidded ''microchip''. This method comprises carrying out desired electrophoretic separation of a liquid sample of an analyte utilizing a flow passage provided in a lidded microchip, and then freezing a water solvent contained in the liquid sample, which has been subjected to the electrophoretic separation, held within the flow passage, lifting the end of a lid part hermetically sealing the upper surface of a grooved flow passage provided on a substrate part at a predetermined speed while maintaining the whole sample subjected to the electrophoretic separation in the frozen state, and separating and removing the lid part from the substrate part under such conditions that maintain flexure having a predetermined radius of curvature.

Description

The automatic sample disposal route and the automatic sample treatment facility that are used for the microchip with sealing lid of bioanalysis
Technical field
The present invention relates to be used under a kind of situation of microchip of the lid sealing that is used for bioanalysis in use at the automatic processing method of the sample to be analyzed of the microchip of lid sealing and according to the automatic sample treatment facility of this method and the biological substance analytical equipment of using the automatic sample disposal route.More specifically, relate to and a kind ofly be used for automatically operation removing the method for cover seal and then handling the sample to be analyzed of the microchip that seals at lid, and the automatic sample treatment facility that is suitable for automatic processing method.
Background technology
For the sample that comprises biological substance, when wanting identification packets to be contained in any protein in the sample or nucleic acid substances, use various electrophoretic techniquess.Special under the very little situation of the absolute magnitude of sample, the capillary electrophoresis method that wherein carries out electrophoresis in having the kapillary of small pore size is widely used as electrophoretic techniques very much, and this technology is for using sample very in a small amount to show good separating property.A kind of like this technology has been proposed, wherein replace having the very kapillary of small pore size, the channel shaped tunnel-shaped of arranging passage with required plane pattern is formed on the substrate, the channel width and the degree of depth for the channel cross-section shape are set at 100 μ m or littler, and the lid seal that plays the lid effect on these passages is arranged in the described passage, makes the effect of channel shaped passage partly be used as capillary space (patent documentation 1).Be provided with this substrate and the combination that is provided with to be scheduled to each other of Qi Gai seal of the passage that can be used as the capillary space use, and together adhering to each other, common as covering " microchip " that seals.When in " microchip " of this lid sealing, carrying out electrophoresis, because nature difference such as electrophoretic velocity, the a plurality of protein and in the nucleic acid substances each that are included in the sample are all separated from one another along passage, so just show a plurality of points (spot), all corresponding every kind of material of each point.Can adopt wherein a plurality of channel shaped passages to be formed on this improved procedure that covers in " microchip " that seals, to design shape with a plurality of passage aisles (electrophoresis path), in the case, different passage aisles (electrophoresis path) each other the state of physical separation realize by the end face that sealing has each the channel shaped passage that covers seal.
After the operation of the electrophoretic separation that is used to be equivalent to Capillary Electrophoresis,, carry out measuring operation to detect along the position of the point of channel separation by utilizing " microchip " of lid sealing.For example, can in the multiple proteins of electrophoretic separation and the nucleic acid substances every kind be set in advance with mark, and detect this mark with the position of location along the point of channel separation.More specifically, proposed a kind of device that is used for the microchip electrophoresis, wherein the form of microchip electrophoresis is that fluorescence labeling uses marking with optical detection point (patent documentation 1:JP 10-246721A).This microchip electrophoretic apparatus by as exciting light source and be used for the fluorescence detector of fluorescently-labeled optical detection this system, be used for as the microchip mobile system of determining the optical detection position along passage, be used to spray electrophoresis liquid and with sample spray the electrophoresis liquid injection system and the sample spraying system of each passage of microchip into, the cpu motherboard that is used for the supply unit of electrophoretic separation and is used to control its action is formed.In addition, be used for this method that the fluorescence labeling of mark carries out optical detection in order to use by utilization, by (for example by light transmissive material, clear glass) form the channel shaped passage in the substrate of making and make " microchip " of lid sealing, and the base component that is used to cover sealing is provided with the electrode that is used for when electrophoresis bias voltage being applied to each channel shaped passage is installed.Behind electrophoresis, each the channel shaped passage that wherein keeps liquid is carried out a little detection.
A kind of equipment has been proposed, wherein after the operation of the electrophoretic separation that is equivalent to capillary isoelectric focusing, " microchip " of adding a cover by utilization, the locational information of point and for along the molecular weight of the protein of its channel separation by using MALDI-MS (ground substance assistant laser parsings/ionization massspectrum method) collection (people such as non-patent literature 1:Michelle L.-S.Mok, Analyst, Vol.129,109-110 (2004), patent documentation 2:WO 03/071263 A1).For fear of by following the liquid in the high pressure heating microchannel that isoelectric focusing applies and the combination evaporator of solvent, the structure that has is for this cools off on thermoelectric (al) cooler with the control temperature with " microchip ", and the end face of each channel shaped passage is closely covered by utilizing the lid sealing.After finishing electrophoretic separation operation, remove lid, and by heated substrates or place it in the vacuum with the rapid solvent evaporation in each channel shaped passage so that at each some place dry with solidify the protein that separates.In the protein that separates remains on state on the microchip, the host material that is fit to is added in this channel shaped passage, then, carry out MALDI-MS along passage and measure to detect each point.
Form one with lid seal and substrate portion and therefore cover under the situation that this form that seal can not be peeled off makes at " microchip " of lid sealing, traditional method capillaceous is identical with using, after electrophoretic separation, (for example need utilize drive unit, pump) taking out the material that separates in passage avoids mixing once more simultaneously, and it is provided for various quality analyses (people such as non-patent literature 2:Daria Peterson, " A New Approach for Fabricating a Zero Dead VolumeElectrospray Tip for Non-Aqueous Microchip CE-MS ", Micro Total AnalysisSystems 2002, Vol.2, pp.691-693 (2002)).In addition, after being taken out, be passed under the situation of single sample retainer, except MALDI-MS, also use EFI to spill ionization mass spectrum determination method (ESI-MS) as mass analysis method by the passage from " microchip " in the individual samples of separating.
Patent documentation 1:JP 10-246721 A
Patent documentation 2:WO 03/071263 A1
People such as non-patent literature 1:Michelle L.-S.Mok, Analyst, Vol.129,109-110 (2004)
People such as non-patent literature 2:Daria Peterson, " A New Approach for Fabricating aZero Dead Volume Electrospray Tip for Non-Aqueous Microchip CE-MS ", Micro Total Analysis Systems 2002, Vol.2, pp.691-693 (2002)
Summary of the invention
The problem that the present invention solves
For the range of application of " microchip " that further expand lid sealing, under a plurality of channel shaped tunnel-shaped are formed in " microchip " of lid sealing with the situation of making out the shape with a plurality of passage aisles (electrophoresis path), need have following two kinds of functions:
Different passage aisle (electrophoresis path) can be by having each the channel shaped passage that covers seal end face physical separation each other; And
On the other hand, after finishing the operation that is used for electrophoretic separation, the lid sealing can separate with baseplate part at an easy rate, then, can be used for the operation of the further analysis of separating base plate at each passage aisle (that is the passage that, is used for electrophoresis).
When the structure that the lid seal of " microchip " of the end face that obtains to be formed at the channel shaped passage in the baseplate part by being used to cover sealing is closely covered, the passage of this tight seal is applicable to the operation of the electrophoretic separation that is equivalent to use conventional electrophoretic method capillaceous.As the mode that constitutes this tight seal passage, need be by utilizing heated sealant or adhesive linkage will form the end face of baseplate part of channel shaped passage and the bottom surface of covering sealing is placed to more firm coherent condition.On the other hand, obtaining under the situation of secure adhesion state more, covering sealing, external force is being applied to covers sealing in order between the bottom surface of the end face of baseplate part and lid sealing, to cause to peel off and then remove, so that force the peel-off covers sealing, but this process may cause small mechanical vibration.These small mechanical vibration may impel the mixing in the liquid that is present in the channel shaped passage, therefore, for example, may cause the diffusion again that is activated at the target substance that separates as narrower point in the channel shaped passage.In addition, during sealing is covered in the spended time removal, when also causing the generation diffusion, with the diffusion again that in the channel shaped passage, occurs to a certain extent as the target substance of narrower some separation owing to the concentration gradient in the liquid.
In addition, when the passage in " microchip " that be formed at the lid sealing by utilization carries out electrophoretic separation, except be formed at baseplate part in the wall of channel shaped passage contact, will never contact with the bottom surface of the lid sealing of the end face of hermetic sealing substrate portion for liquid.In the case, when peeling off and remove when covering sealing, the moisture of the fraction liquid that contacts with the bottom surface of lid sealing by liquid caused to adhere to the bottom surface of covering sealing.After this, this a spot of liquid can go downwards to the bottom surface of covering sealing, forms the drop that gathers, and then, drop splashes into passage once more.In the zone that these drops that gather drip once more, if drop that has fallen and herein liquid mixing then will enter the state that is exposed to " outer interface " of the virtual condition that is different from separation.
In " microchip " of lid sealing, the end face that is formed at the channel shaped passage in the baseplate part that covers " microchip " that seal is by closely being covered with the lid both seals, therefore, when being formed in " microchip ", a plurality of channel shaped tunnel-shaped go up when being made into the have a plurality of passage aisles shape of (passage that is used for electrophoresis), different passage aisle (passage that is used for electrophoresis) is placed to and makes its state of physical separation each other, thereby avoids causing the infiltration of the liquid between passage aisle (passage that is used for electrophoresis) and the seepage of liquid well, the evaporation of solvent and the intrusion of exterior materials.Yet, need further to suppress as owing to peel off the phenomenon of " the inherence pollution " that cause with the diffusion again of removing the target substance that separates that the action of cover sealing causes and by the seldom quantity of fluid that adheres to the bottom surface of cover sealing.
In addition, cover being operating as manually of sealing and carry out if be used to peel off and remove, then the length of spended time is along with the fluctuation of workman's technical merit, and from obtaining the viewpoint of high duplication, need realize the operation of peeling off and removing of covering sealing by automated procedure.
The present invention will address the above problem, therefore the purpose of this invention is to provide a kind of sample treatment, and based on the automatic sample treatment facility of this automatic sample disposal route, in the method, after " microchip " with sample liquids utilization lid sealing to be analyzed is used for the operation of electrophoretic separation, adhere to and be fixed to the peeling off and remove operation and can carry out high-caliber repetition of lid sealing of end face of the baseplate part of " microchip " of forming the lid sealing, suppress the diffusion again of the target substance that separates simultaneously and by adhering to " the inherent pollution " that the seldom quantity of fluid that cover the sealing bottom surface causes by utilizing automatic equipment.
The means that are used to deal with problems
The present invention is devoted to thorough research addressing the above problem, and obtains following series and find.
At first, the researchist notices, two phenomenons of " diffusion again of target substance " are because by peeling off and removing that the milli machine that action caused that cover sealing vibrates the mixing of the liquid cause and the dispersion of the concentration that caused by the concentration gradient of the target substance that separates, even after finishing the electrophoretic separation operation, these two kinds of phenomenons also are easy to occur under material keeps as the situation that is formed at the solution in the passage that covers in " microchip " that seals.Therefore, the inventor finds the solid state shape of migration in be placed on material is difficult to, rather than remains on solution state and will can prevent two kinds of phenomenons of " diffusion again of target substance " substantially.Particularly, find after finishing the electrophoretic separation operation, carrying out fast, cooling remains on the operation of the solution in this passage with the freezing hydrosolvent that is included in wherein, then, when keeping this freezing state, be used to peel off and remove the operation of cover sealing, can avoid thus since the mixing of the liquid that the milli machine vibration causes with since the concentration that the concentration gradient of the target substance that separates causes spread.
In addition, in peeling off and remove the step of covering sealing, when peeling off, between the bottom surface of the end face of baseplate part and lid sealing, cause instantaneous narrow gap inevitably.Remain under the situation of permeating then in these passages in narrow gap at part solution, may cause sometimes because the capillary effect at the interface between the bottom surface of the end face of the baseplate part that peel off on the edge and lid sealing increases leakage.As a result, sizable possibility is arranged because the local penetration of the liquid between the adjacent passage aisle (passage that is used for electrophoresis) causes appearance " pollution mutually ".The sample of the electrophoretic separation in the passage in being formed at " microchip " remains under the situation of freezing state, in peeling off and remove the step of covering sealing, when the narrow gap of instantaneous appearance between the bottom surface of the end face of baseplate part and lid sealing, just essence is prevented the phenomenon of the local penetration that remains on the liquid in these passages.Therefore, can find, during the instantaneous narrow gap between the bottom surface of the end face of baseplate part and lid sealing and peeling off and removing in the spacing and width in the instantaneous narrow gap in the step of covering sealing, can remove any restriction substantially.
At last, phenomenon for " the inherent pollution " that cause by the small amount of liquid that adheres to the bottom surface of covering sealing, the inventor also finds, after carrying out the freezing operation that is included in hydrosolvent wherein in advance, keeping under the situation of this freezing state, any problem no longer occurring at the moisture of the lip-deep liquid of the bottom surface of lid sealing.On the other hand, contact with each other, must under this state, peel off and remove and cover sealing on the surface of the bottom surface of the end face of the sample of freezing state and lid sealing, so as to allow under freezing state sample satisfactorily with the surface isolation of the bottom surface of lid sealing.The fragment that the bonding strength on the surface of the bottom surface of sample under freezing state and lid sealing can not cause the part to leave the sample of freezing state is stayed under the lip-deep situation of the bottom surface of covering sealing, need cover peeling off and removing of sealing by selection mode.
The inventor after deliberation no matter under what environment, the fragment of the sample separation under part and the freezing state will be stayed on the surface of the bottom surface of covering sealing.After this, the inventor finds, peeling off, promptly in peeling off and remove the step of covering sealing, the narrow gap portion of instantaneous appearance between the bottom surface of the end face of the sample of freezing state and lid sealing, the surperficial position contacting place of the bottom surface of the end face of the sample under freezing state and lid sealing, the lid sealing temporarily is in the state with the bending of certain curvature radius R; And if select radius of curvature R less than certain threshold R Eq1(R＜R Eq1) state, then can not occur any fragment that leaves the sample of freezing state again and stay and cover on the sealing.Further disclosed according to the Young modulus E that constitutes material cover sealing, freezing state under sample and cover the bonding strength p of per unit area of the bottom surface of sealing 1Determine critical value R with the shear resistance level Eq1, be higher than critical value R Eq1Will the fragment of the sample of freezing state appears.
According to above-mentioned discovery, following situation that the inventor is verified, thus finish the present invention:
After the passage in the microchip that is formed at the lid sealing by utilization is used for the required electrophoretic separation operation of fluid sample to be analyzed,
When keeping sample by electrophoretic separation in the channel shaped passage in being formed at baseplate part in the freezing state that continues, the end face that up to the present adheres to and be fixed to the baseplate part in the microchip of lid sealing is formed at the operation that the lid sealing of the end face of the channel shaped passage in the baseplate part separates with sealing can be undertaken by following steps:
The fluid sample that experiences electrophoretic separation and remain in the passage is passed through the freezing operation that comprises hydrosolvent within it; And
The operation that the lid sealing that seals the end face that is formed at the channel shaped passage the baseplate part was peeled off and removed to execution from baseplate part, the sample that keeps simultaneously having experienced electrophoretic separation in passage is in lasting freezing state; And further
Can carry out above-mentioned series operation automatically.
Therefore, the automatic sample disposal route according to the microchip of the lid sealing that is used for bioanalysis according to the present invention is:
A kind ofly treat to be used for the method that automatic processing has been experienced electrophoretic separation and remained on the fluid sample of passage the biological fluid sample of analyzing is formed at the operation of the required electrophoretic separation of passage experience in the microchip of lid sealing by utilization after, it is characterized in that when making:
The structure that the microchip of described lid sealing has is, the lid sealing that is formed at the end face of channel shaped passage in its baseplate part and hermetic sealing substrate portion has obtained to bond to together state with predetermined set, make that the end face of baseplate part is closely bonding each other with the bottom surface of lid sealing
After the passage in the microchip that is formed at the lid sealing by utilization is finished the required electrophoretic separation operation of fluid sample to be analyzed, said method comprising the steps of:
Cooling step, wherein by electrophoretic separation and remain on the freezing operation that comprises hydrosolvent within it of baseplate part of the fluid sample experience microchip by cooling off described lid sealing in the passage, to realize being in freezing point or subfreezing predetermined low-temperature condition;
Peel off the step of covering sealing, wherein keep being cooled to described predetermined low temperature level by the baseplate part with the microchip of described lid sealing simultaneously, the sample that keeps having experienced electrophoretic separation is in lasting freezing state in passage,
Peeling off and remove the operation of covering sealing from baseplate part is undertaken by external force being applied to the end of covering sealing, so that discharge the bonding strength that end face that has made baseplate part and the bottom surface of covering sealing closely contacted and obtained with predetermined arrangement adhering state, thereby the bottom surface of the lid sealing that peels off from the end face of baseplate part is simultaneously with respect to predetermined critical value R Eq1, the radius of curvature R that maintenance is illustrated in the local bending of the borderline lid sealing of peeling off keeps less than described critical value R Eq1(R＜R Eq1) state; And
The step of dividing the sealing of uncapping, wherein after described strip step finishes, in the microchip of lid sealing, be separated by the lid sealing that has separated that is adhesively fixed that discharges on it with the end face of baseplate part, then, make the lid sealing experience of separation be used for its carry away from and the operation that keeps so that obtain to make the surface of the sample of the electrophoretic separation under the freezing state that continues to be exposed to state in the channel shaped passage that is formed in the baseplate part; And
A series of these steps realize automatically.
In addition, the automatic sample disposal route of the microchip that the lid that is used for bioanalysis according to the present invention seals also can have this structure, and method is characterised in that:
When finish be used to remove the process of covering sealing after,
Method also comprises:
Be used for freeze-drying and fixing step, wherein continuing sample that freezing state remains on the electrophoretic separation in the channel shaped passage that is formed in the baseplate part by freeze-drying, each component substances that separates with each the some place of the point on will the described passage in being formed at baseplate part is fixed on the relevant point with the form of lyophilized substance.
In addition, the automatic sample treatment facility according to the microchip of the lid sealing that is used for bioanalysis according to the present invention is:
A kind of being used for is characterized in that treating the required electrophoretic separation operation back of the biological fluid sample experience of analyzing, experience electrophoretic separation and remain on the equipment that the fluid sample in the passage is handled automatically will making by the passage that utilization be formed at the microchip of lid sealing:
The structure that the microchip of described lid sealing has is, the lid sealing that wherein is formed at the end face of channel shaped passage in its baseplate part and hermetic sealing substrate portion has obtained to bond to the state that makes that together the bottom surface of the end face of baseplate part and lid sealing is closely adhered to each other with predetermined arrangement
Described equipment comprises the following system that is provided for covering the microchip of sealing, and the action required of the electrophoretic separation of fluid sample wherein to be analyzed is finished by the passage that utilization is formed in the microchip of lid sealing:
The system that is used for refrigerating base board portion, this system are suitable for the arrangement installation that contact with the baseplate part of the microchip of described lid sealing;
The control module that is used for refrigerating system, by the system that is used for refrigerating base board portion of installing with the arrangement that contacts with baseplate part, this unit can remain on and is under freezing point or the subfreezing predetermined low temperature level state by being cooled to major general's baseplate part;
The system that is used for fixing baseplate part, this system can fix the baseplate part of the microchip of described lid sealing with the arrangement that contacts with the described system that is used for refrigerating base board portion;
Be used to apply the system of external force, the function that this system has is, the arrangement of fixing by the described system that is used for fixing baseplate part with baseplate part, on the end of lid sealing, apply the external force of direction, so that discharge end face that has made baseplate part and the tight bonding strength that contacts and obtain adhering state with predetermined arrangement of the bottom surface of covering sealing with the end face that is basically perpendicular to baseplate part;
Be used to carry the system of the end of covering sealing, this system can with by the described system that is used to apply external force external force is applied on the end of covering sealing synchronously, on the direction of the Contact Boundary between the bottom surface of end face that is basically perpendicular to baseplate part and lid sealing, carry the end of covering sealing;
Be used to control the system of the speed of carrying the end of covering sealing, the function that this system has is covered the transporting velocity of the end of sealing for control, make the process that peels off from the end face of baseplate part in the bottom surface that will cover sealing by the system that utilizes with the system that is used for carrying the end of covering sealing of on the end of described lid sealing, synchronousing working and be used to apply external force, with respect to the critical value R that is scheduled to Eq1, the radius of curvature R of the local bending of the borderline lid sealing that expression is peeled off remains on radius of curvature R and keeps less than described critical value R Eq1(R＜R Eq1) state under;
Be used for the separately system of the lid sealing of separation, the function that this system has is, after will covering the EO that the end face of sealing from baseplate part peel off, the lid sealing that system's maintenance is adhesively fixed and separates with the end face of baseplate part by release, and with of the end face conveying of described lid sealing, so that expose the channel shaped passage that is formed in the baseplate part away from baseplate part; And
Described equipment comprises also and is used to control its automatic operated system that the function that this system has is that the action of each system that realizes the series operation is realized according to the predefined procedure Automatic Program.
In addition, can have this structure, being characterized as of this equipment according to the automatic sample treatment facility of the microchip of the lid sealing that is used for bioanalysis according to the present invention:
Except above-mentioned each system,
Described equipment also comprises,
Be used for freeze-drying and fixing system, utilize this system, the sample that remains on the electrophoretic separation in the channel shaped passage that is formed in the baseplate part with the freezing state that continues with following state by freeze-drying, described state for be used for by utilization branchs uncap lid sealing that the system of sealing will separate away from the end face conveying of baseplate part to expose the channel shaped passage that is formed at baseplate part, make each component substances of each some place separation of the point on the described passage in being formed at baseplate part be fixed on the relevant point with the form of lyophilized substance.
In addition, the present invention also is provided for the invention of the method for biological sample analysis, wherein, by using automatic sample disposal route according to the microchip of the lid sealing that is used for bioanalysis with said structure according to the present invention, after the microchip that utilizes the lid sealing is finished the operation of electrophoretic separation, peel off and remove the lid sealing of the end face that passes through the tight covered substrate of sealing portion, then, the electrophoretic separation sample is used for the operation of quality analysis, this sample remains in the channel shaped passage that is formed in the baseplate part with the freezing state that continues, and sample surfaces exposes by processing.
Therefore, biological sample analysis method according to the present invention is:
A kind of method that is used for analysis of biological samples, in described method, after the passage in the microchip that is formed at the lid sealing by utilization will treat that the biological fluid sample of analyzing is through required electrophoretic separation operation, the component substances that separates for the point on described passage carries out the quality analysis of the component substances of a separation, these materials will be included in the fluid sample of the electrophoretic separation on the passage that remains in the microchip that is formed at the lid sealing, it is characterized in that:
The structure that the microchip of described lid sealing has is: the lid sealing that is formed at the end face of channel shaped passage in its baseplate part and hermetic sealing substrate portion has obtained the state that bonds together with predetermined arrangement, make the end face of baseplate part and the bottom surface of lid sealing be closely adhered to each other with
Described method comprises:
Collect step, wherein, after the passage in the microchip that is formed at the lid sealing by utilization is finished fluid sample to be analyzed required electrophoretic separation operation,
Peel off and remove lid sealing according to the automatic sample disposal route of the microchip with sealing lid that is used for bioanalysis of the present invention by the end face that seals tight covered substrate portion with aforesaid structure, then,
Collect the sample of electrophoretic separation, the sample of electrophoretic separation remains in the channel shaped passage that is formed in the baseplate part to continue freezing state, thereby exposes the surface of the sample of electrophoretic separation;
Be used for freeze-drying and fixing step, wherein continuing sample that freezing state remains on the electrophoretic separation in the channel shaped passage that is formed in the baseplate part by freeze-drying, each component substances that separates with each the some place of the point on will the described passage in being formed at baseplate part is fixed on the relevant point with the form of lyophilized substance;
Be used to provide the step of matrixing agent (matrixing agent), wherein be used for the matrixing agent that MALDI-MS analyzes and be applied to the channel shaped passage that is formed at baseplate part, described matrixing agent is provided to the component substances of form with the lyophilized substance electrophoretic separation on being fixed to a little;
Be used for the step that MALDI-MS analyzes, wherein by utilizing described matrixing agent to carry out the MALDI-MS analysis operation along the channel shaped passage that is formed in the baseplate part, molecular weight information with the ionic species that obtains to obtain by component substances through electrophoresis, be fixed on the reference point through the component substances of electrophoresis form with lyophilized substance, and the positional information of the point of the molecular weight information of acquisition demonstration ionic species, and
The step that is used for data analysis, wherein show that according to being used to of obtaining the positional information of point of the molecular weight information of ionic species (ionspecies) specifies the electrophoresis exponential quantity corresponding to point, then this information is changed into the concrete electrophoresis exponential quantity of appointment and in the combination of the molecular weight information of the ionic species of measuring along the reference point place of channel shaped channel location, described electrophoresis exponential quantity and information are inferred from the component substances that is included in the fluid sample to be analyzed.
Effect of the present invention
By utilizing according to the automatic sample disposal route of the microchip of the lid sealing that is used for bioanalysis according to the present invention and the automatic sample treatment facility of microchip that is used for the lid sealing of bioanalysis, after carrying out the electrophoretic separation operation of sample liquids to be analyzed by " microchip " that utilizes the lid sealing, can be used to peel off and remove the operation of the lid sealing of the end face that adheres to and be fixed to baseplate part with high-caliber repeatability automatically, described baseplate part and described lid sealing are formed " microchip " of lid sealing, limit the diffusion again of the target substance that separates simultaneously and owing to adhere to " inherent pollution " phenomenon that the small amount of liquid of the bottom surface of covering sealing causes.In addition, be used for carrying out automatically with high level repeatability peel off and remove the operation of covering sealing after, by utilizing the sample that has experienced electrophoretic separation, also can be used for the operation of the specimen preparation of further analysis (for example, quality analysis) automatically in advance.Therefore, even the quantity of the sample liquids to be analyzed of process electrophoretic separation becomes big, by using the high level repeatability that the present invention also can procurement process, the sample for the separation that is provided for further analyzing in this process carries out sample preparation to the sample that experiences electrophoretic separation.
Description of drawings
Fig. 1 is the sketch that the problem that solves by the present invention schematically is described;
Fig. 2 is the sketch that the example of the passage in the microchip that is used for the present invention schematically is described;
Fig. 3 is the sketch of example that the microchip structure of the lid sealing that is used for the present invention schematically is described;
Fig. 4 is the sketch of another example that the microchip structure of the lid sealing that is used for the present invention schematically is described;
Fig. 5 is the sketch of example that the structure of the stripping system that is used for the lid sealing that can use at automatic sample treatment facility according to the present invention schematically is described, and the principle of work of being utilized in the stripping system in first exemplary embodiments has been described;
Fig. 6 is the sketch of example that the structure of the stripping system that is used for the lid sealing that can use at automatic sample treatment facility according to the present invention schematically is described, and the principle of work of being utilized in the stripping system in second exemplary embodiments has been described;
Fig. 7 is the sketch of example that the structure of the stripping system that is used for the lid sealing that can use at automatic sample treatment facility according to the present invention schematically is described, and the principle of work of being utilized in the stripping system in the 3rd exemplary embodiments has been described;
Fig. 8 is the sketch of example that the structure of the stripping system that is used for the lid sealing that can use at automatic sample treatment facility according to the present invention schematically is described, and the principle of work of being utilized in the stripping system in the 4th exemplary embodiments has been described;
Fig. 9 is the sketch of example that the structure of the stripping system that is used for the lid sealing that can use at automatic sample treatment facility according to the present invention schematically is described, and the principle of work of being utilized in the stripping system in the 5th exemplary embodiments has been described;
Figure 10 is the sketch of example that the structure of the stripping system that is used for the lid sealing that can use at automatic sample treatment facility according to the present invention schematically is described, and the principle of work of being utilized in the stripping system in the 6th exemplary embodiments has been described;
Figure 11 is the sketch of example that the structure of the stripping system that is used for the lid sealing that can use at automatic sample treatment facility according to the present invention schematically is described, and the principle of work of being utilized in the stripping system in the 7th exemplary embodiments has been described; And
Figure 12 is the sketch that another example of the passage in the microchip that is used for the present invention schematically is described.Below use label in the drawings to have following implication respectively.
101: the plane cover base part
102: the adhering resin rete
103: baseplate part
105a, 105b, 105c, 105d: liquid reservoir
107a: injection channel
107b: split tunnel
110: the electrode terminal fixture
112: the microchip of lid sealing
113: the lid sealing
Embodiment
To illustrate in greater detail the present invention below.
The sample of handling in automatic sample disposal route according to the present invention is the electrophoretic fluid sample, be formed at the required described electrophoretic fluid sample of electrophoresis operation preparation of fluid sample experience that the passage that covers in the microchip that seals is used in bioanalysis by utilization, thereby make the multiple material location separated from one another that is included in the fluid sample, so that form point along channel location.When the fluid sample of electrophoretic separation has finished in predetermined electrophoretic separation, keep liquid condition in the passage in the microchip that is formed at the lid sealing.When the fluid sample of electrophoretic separation during, must experience the specimen preparation that next basis is handled the bioassay technique of application as the sample in the ensuing bioanalysis.
For example, be under the situation of quality analysis at ensuing bioassay technique, must make separation locate the processing that is used for drying with the material experience that forms point along passage at once, from this material, to remove solvent composition.In this process, must can prevent to separate the location by utilization and be used for dry processing with the method that each material that forms point along passage is mixed with each other.The form that is used to handle according to automatic sample disposal route of the present invention and automatic sample treatment facility is, the fluid sample that wherein omits the electrophoretic separation in the passage in the microchip that will be formed at the lid sealing takes out the processing operation of this passage, therefore, the solvent composition that comprises in the described sample is by being removed sample being remained on point evaporate in the state in the passage.
(fluid sample of pending electrophoretic separation)
To at first be illustrated as the fluid sample of the electrophoretic separation of the target of handling by automatic sample disposal route according to the present invention and automatic sample treatment facility below.
Be formed under the situation of the passage in the microchip that covers sealing in utilization, can use the electrophoretic separation that is equivalent to traditional Capillary Electrophoresis.Particularly, in being included in fluid sample, treat that the biological biomolecule of analyzing is under the situation of protein, can use the isoelectric focusing of the different protein of the differential liberation of the isoelectric point by utilizing every kind of protein indication or the difference of the mobility speed (phoretic velocity) that obtains by the difference of utilizing by molecular weight makes protein swimming separation separated from one another.The biomolecule for the treatment of biological analysis in being included in fluid sample is under the situation of nucleic acid molecules, can use swimming to separate by utilizing difference on the length of basis (base) (that is the difference of the mobility speed that obtains by the difference of molecular weight) to separate the different IPs acid molecule.
In the case, being formed at the flat shape of the passage in the microchip that covers sealing itself, the layout of passage and the length of passage suitably selects according to the method for electrophoretic separation of using.For example, can select the channel architecture with flat shape shown in Figure 2.In channel architecture shown in Figure 2, the injection channel 107a that is used for the split tunnel 107b that separates by isoelectric focusing and is used for biomolecule to be focused on (for example, protein) is incorporated into path 10 7b is provided on the end face of baseplate part 103.At place, the two ends of split tunnel 107b, form liquid reservoir 105d and 105c, be used to provide the acidity of pH gradient and akaline liquid all to be introduced into liquid reservoir 105d and 105c, the terminal that is used for applying the electrode of electric field between it is inserted into described liquid reservoir.The place, two ends of injection channel 107a also forms liquid reservoir 105a and 105b.The electrode that is used to apply electric field also is inserted into liquid reservoir 105a and 105b, is used for the electric field of migration of the protein of injection channel 107a with generation.
Under the situation of the electrophoretic separation of using as isoelectric focusing, can select to omit the channel architecture of the injection channel 107a in the channel architecture shown in Figure 2, and the split tunnel 107b in the separation that only is provided for being undertaken by isoelectric focusing.Figure 12 has shown the example of the channel architecture that only is provided with the split tunnel 107b that is used for the separation undertaken by isoelectric focusing.Liquid reservoir 105d and 105c are formed at the two ends of the split tunnel 107b in the end face that is installed in baseplate part 103, and the acidity and the akaline liquid that are used for producing the pH gradient all are introduced in these liquid reservoirs 105d and 105c.Insertion is used to apply the electrode terminal of electric field, is used for the electric field of migration of the protein of split tunnel 107b with generation.In addition, although split tunnel 107b shown in Figure 12 be shaped as the single channel structure, it can be extended to wherein a plurality of channel shaped passages and be arranged on multi-channel type microchip in the end face of baseplate part 103.
(structure of the microchip of lid sealing and the electrophoresis operation that utilizes this structure)
The microchip of lid sealing is made of baseplate part 103 and lid sealing 113, forms the channel shaped passage with required flat shape in the end face of wherein said baseplate part, and the end face of this channel shaped passage is closely covered through sealing by described lid sealing.In addition, the hole that is used for the liquid injection is formed at covers sealing 113, and the liquid reservoir with the end that is arranged on the channel shaped passage is complementary respectively, the end face of simultaneously complete covering groove shape passage.Lid sealing 113 by the plane cover base part 101 of function with the physical strength that keeps covering sealing 113 and the adhering resin rete 102 that on its bottom surface portions, is used to bond to the end face of baseplate part 103 constitute.Be installed in Kong Douyu liquid reservoir 105d and 105c and liquid reservoir 105a and the 105b aligning that liquid sprays that be used in plane cover base part 101 and the adhering resin rete 102.In addition, also use when the electrode terminal that is used to apply electric field is inserted into liquid reservoir 105d and 105c and liquid reservoir 105a and 105b in the hole that liquid sprays of being used for that is installed in plane cover base part 101 and the adhering resin rete 102.In addition, in some cases, also can make plane cover base part 101 and be used for its bottom surface is bonded to the adhering resin rete 102 of end face of the baseplate part 103 of same material.When identical materials was used for two elements, these two elements can manufacture the one form in advance.
The hole that liquid sprays that is used in order to be used for applying the electrode terminal connection of electric field and to be fixed to plane cover base part 101 is provided to plane cover base part 101 with electrode terminal fixture 110 in advance.Before the electrophoresis operation, the electrode terminal that is used to apply electric field can be by utilizing electrode terminal fixture 110 fixing, and transferring to the stage that automatic sample is handled from the electrophoresis EO, the electrode terminal that is used to apply electric field is split from electrode terminal fixture 110.This lid base part 101 and electrode terminal fixture 110 can be made and assemble or be made by identical materials by different materials, and under situation about being made by identical materials, lid base part 101 and electrode terminal fixture 110 can manufacture the one form in advance.By utilizing the electrode terminal connection/split system of mutual alignment of a plurality of electrode terminals that is used to apply electric field pre-determined use, when the microchip fixed system that utilizes electrophoresis equipment the precalculated position arrange and the microchip of fixed cap sealing after, can realize following electrophoresis to operate and be used to applying these connections and the fractured operation of the electrode terminal of electric field.Certainly, also can connect and split the microchip of electrode terminal and fixed cap sealing by manual operation, and can make the microchip fixed system that is provided with electrophoresis equipment be connected with electrode terminal/split system constitutes automatic operation format.Owing to need especially in this invention carry out a series of sample preparation automatically by the microchip itself that remains on its position and operate, preferably has the form of the operation of the microchip that the connection of carrying out electrode terminal automatically and fractured operation and fixed cap seal when finishing electrophoresis operation back.
In addition, in example structure shown in Figure 3, equipment electrode terminal fixture 110, and also to constitute the form fixed electorde terminal fixture 110 of the sidewall sections that is installed in the hole that is used for the liquid injection in the plane cover base part 101, and can be as another structure shown in Figure 4, select equipment electrode terminal fixture 110 and to be connected to the structure of the form fixed electorde terminal fixture 110 that is installed in the upper end that is used for the hole that liquid sprays in the plane cover base part 101.
Baseplate part 103 and lid sealing 113 are used for separately according to it that liquid sprays and the position in the hole of liquid reservoir is aligned with each other, cover the structure that sealing 113 sealings come the end face of tight covering groove shape path 10 7a to constitute by end face that makes baseplate part 103 and bottom surface (that is, the adhering resin rete 102) utilization adhering to each other of covering sealing 113.For laminating flat lid base part 101 and adhering resin rete 102, use the bond tool that shows the high adherence energy; When in lid sealing 113 will the step in the back, being stripped from and removing, select it to peel off the form of on the end face of baseplate part 103 and the bonding plane between the adhering resin rete 102, carrying out.Therefore, to show sufficient adhesion strength at the end face of baseplate part 103 and the bonding plane between the adhering resin rete 102, to obtain enough adhering state closely, the problem that may leak or leach from the channel shaped path 10 7a the end face that is formed at baseplate part 103 with the swimming liquid of avoiding being filled in the passage, but can peel off along this bonding plane by applying predetermined external force.
When using according to automatic sample disposal route of the present invention and automatic sample treatment facility, the high-adhesive-strength that preferably utilizes adhering resin rete 102 itself keeps adhering state closely between the end face of baseplate part 103 and adhering resin rete 102.Yet, in the bonding strengths that reduce adhering resin rete 102 itself and should be than low-intensity with under the pattern that keeps baseplate part 103 and lid sealing 113 and closely bond, also can between the end face of baseplate part 103 and adhering resin rete 102, keep the state that closely bonds by applying the external force load compensation.Apply the means of external force as being used to, can adopt the form (that is load application system) of imposed load on the end face of lid sealing 113 to keep baseplate part 103 and lid sealing 113 to closely bond.It is desirable to select the pattern that load wherein can basically identical ground dispersion on the whole bonding plane between baseplate part 103 and the lid sealing 113 for this load application system.In addition, be similar to the microchip fixed system and be connected with electrode terminal/split system, be preferred for peeling off and remove the operation of covering sealing 113 constituting the pattern that to operate automatically, the load that applies with removal.For example, the load application system is connected with electrode terminal/and split system can form as one, and when by behind the load application system imposed load, equips electrode terminal by electrode terminal connections/split system.
For the end face of baseplate part 103, material is selected the material of the processing accuracy that can obtain to expect when making meticulous channel shaped path 10 7 within it when the processing of carrying out above-mentioned fine structure.According to the electrophoresis method that uses, the cross sectional shape of channel shaped passage to be formed is chosen as channel width (W 1) and channel depth (D 1) scope be that 5 μ m are to 1000 μ m.The meticulous channel shaped passage of this " microchip " is mainly used in the electrophoretic separation operation that the fluid sample that utilizes denier replaces capillary electrophoresis to carry out.Therefore, ideally, select the area of section (D of meticulous channel shaped passage 1* W 1) the same little with interior area of section capillaceous, for example, in the scope of the area of section that is no more than 100 μ m internal diameters.On the other hand, the suitable selector channel degree of depth (D 1)/channel width (W 1) ratio (D 1/ W 1), also to consider the material of baseplate part 103 and the processing accuracy of determining by the fine processing that is used for the channel shaped passage.Usually, because too high ratio (D 1/ W 1) will increase the difficulty of handling, therefore, be chosen in 1/100≤D ideally 1/ W 1Ratio in≤10 the scope.
In addition, when using according to automatic sample disposal route of the present invention and automatic sample treatment facility, the sample that makes electrophoretic separation is subjected to being used for fixing processing in these meticulous channel shaped passages by desivac, be fixed on the reference point with material as freeze-drying, then, the component substances that will separate on described sample is used for the MALDI-MS analysis.In this step, because the pattern of using ionic type to produce by the freeze dried substance on the bottom surface that is present in the channel shaped passage and the opening of ionic type from the end face of channel shaped passage that produces being taken out, so, ideally, usually at D 1/ W 1Select in≤1 the scope.
When using,,, or also can be open top part (W so the cross sectional shape of channel shaped passage also can be rectangle owing to keep keeping the state of electrophoretic separation sample with freezing state according to automatic sample disposal route of the present invention and automatic sample treatment facility 1top) width than the bottom surface (W of groove 1 Bottom) the narrow (W of width 1bottom＞W 1top) trapezoidal, be difficult to make the sample of freezing state to leave like this.
As the material of baseplate part 103, suitably use the material that is applicable to fine processing, for example, quartz, glass or silicon.In addition, the anticipate accuracy of fine processing can be obtained to be undertaken, also PDMS and PMMA can be used by the plastics of the high-insulation that comprises polycarbonate.When electric field applies in the channel shaped passage in the end face that into is formed on baseplate part 103, baseplate part 103 itself need with the swimming fluid insulation in the channel shaped passage, therefore, ideally, use the material of high-insulation, for example, quartz or glass.Use the relatively poor material of insulating property (for example, silicon under) the situation, use insulating coating to be arranged on structure on the inwall of channel shaped passage, with obtain with the channel shaped passage in the electrical isolation of expection of swimming liquid.Alternatively, also can adopt the channel shaped channel part to be formed at the pattern that the silicon dioxide layer on the silicon substrate forms by utilization.
In addition, in the present invention, in the step of peeling off, baseplate part 103 is elastic deformation not only, and covers also elastic deformation of sealing 113, thereby on the boundary member of peeling off flexible structure is set.Therefore, be used for plane cover base part 101 with showing flexible material.Perhaps, lid sealing 113 can have enough thickness to allow to cover sealing 113 elastic deformations.
In the present invention, can stand to have good insulating property, and also have the flexibility of the material that is applicable to plane cover base part 101 such as this material of the processing of installing the hole that is used for the liquid injection within it.For example, preferred use acrylic resin (for example, PMMA (polymethylmethacrylate)) or comprise a kind of material in the polymeric resin material of PDMS (dimethyl silicone polymer), even particularly be easy to handle but the also planarization material of easy fracture (flat material) not of very thin thickness.For the material of the substrate that is used for adhering resin rete 102, for example, use PDMS, comprise a kind of or polyester in the polyolefin (polyorefine) of PTFE (teflon), PP (polypropylene), PE (tygon) and Polyvinylchloride.For adhering resin rete 102, preferably use the elastically deformable material higher than the material that is used for plane cover base part 101.The form that coating with bonding agent of cementability is arranged on the outermost layer of adhering resin rete 102 is more satisfactory.In addition, there is not the coating of bonding agent corresponding to the zone of the end face of the channel shaped passage of this layer, but preferably can be for showing the surface of hydrophobicity and water proofing property.Therefore,, can suitably use material with water proofing property and grease proofness for the substrate of resin film layer 102, for example, such as the fluorine resin of PTFE.
In the microchip of lid sealing itself, the profile of baseplate part 103 is a rectangle, and the profile of the lid sealing 113 of the end face of hermetic sealing substrate portion 103 also is a rectangle.When peeling off and remove when covering sealing 113, for external force being applied to an end that covers sealing 113, the part of protruding from the profile of baseplate part 103 can be at least be provided with towards being used for the end that external force applies.For example, under the situation of the rectangle longer sides of peeling off and remove the selected profile along baseplate part 103 of the direction of covering sealing 113, it is bigger than the longer sides of baseplate part 103 that the profile of lid sealing 113 is made for longer sides.When external force is applied to when covering sealing 113, can set cover sealing 113 the working point in the part of protruding from its longer sides.In addition, when finish peel off and remove cover sealing 113 after, zone when the end by keeping and when carrying the lid sealing that separates to carry out fractured operation, can in described projection, set being used for the lid sealing by the separation of maintenance system support away from the end face of baseplate part.In addition, can also select to use along the longer sides of baseplate part 103 lid sealing 113 than short side direction on the part protruded as when peeling off and remove the pattern that applies the place of external force when covering sealing 113.
(being used for electrophoresis liquid is sprayed the system that into is installed in the passage that covers the microchip that seals)
As mentioned above, for the passage in the microchip that is installed in the lid sealing, suppose its area of section (D 1/ W 1) the same with area of section capillaceous little, but do not constitute the relatively poor rare cases of water wettability of material of the lid sealing 113 of its end face.The kapillary of being made by the material of high-hydrophilic allows electrophoresis liquid to supply to whole kapillary from an end of passage by capillarity, but for the passage in the microchip with relatively poor hydrophilic internal face, liquid injection system must be set, to replace the utilizing electrophoresis liquid of tubule phenomenon to spray.Particularly, the preferred pattern of using is to produce pressure differential between the inside of liquid reservoir that is arranged on the channel end place and passage, and utilize the pressure differential that is caused to be used for forcing the electrophoresis liquid that will supply with from a liquid reservoir to spray stand in channel.
Because the passage itself that is formed in the microchip that covers sealing is closely covered by sealing, so when staying that gas inside is discharged by liquid reservoir and electrophoresis liquid when supplying to other liquid reservoir, because the pressure differential between it, electrophoresis liquid penetrates in the passage.In this step,, stop to spray when electrophoresis liquid during the complete filling passage.Utilize in use under the situation of the electrophoresis liquid spraying technique that pressure differential carries out, utilize following structure can be used for the operation that electrophoresis liquid sprays automatically, in this structure: be used to extract out the suction system of staying gas inside and be connected to a liquid reservoir; Liquid feed system with the little liquid measure that is applicable to the electrophoresis liquid that sprays scheduled volume is connected to another liquid reservoir; In addition, two systems all are connected to the judgement system that determines the end sequential of its injection action automatically.
As the judgement system that automatically determines the end sequential of injection action, for example, can use utilize as following example in order to detect the whether judgement system of the detecting unit of complete filling passage of the electrophoresis liquid that sprays.
When electrophoresis liquid during the complete filling passage, when electrophoresis liquid itself when having the medium of some electric conductivity, the quick change from the state of insulation to the predetermined impedance can observe by the resistance value of monitoring between each passage two ends.Utilize electrophoresis liquid to play this impedance monitoring type detecting unit of conducting medium function by every end equipment, can make the judgement of the occupied state of electrophoresis liquid at each passage.
In addition, electrophoresis liquid is liquid, and the specific inductive capacity of electrophoresis liquid is obviously different with gas dielectric constant.By utilizing this feature can use following detecting unit, in this detecting unit, two electrodes of plane capacitance type are arranged on the two side of each passage, to detect the phenomenon that makes the electric capacity change when electrophoresis liquid penetrates in the space between the electrode.By the detecting unit of equipping this plane capacitance type at every end place of each passage, can make the judgement of the occupied state of electrophoresis liquid.
In addition, when electrophoresis liquid was liquid, as specific inductive capacity, the electrophoresis liquid refractive index was obviously different with the refractive index of gas.For example, under the situation that baseplate part 103 is made by light transmissive material, when electrophoresis liquid covering wall face, the light reflectance at the wall place of the passage in being formed at the end face of described baseplate part changes.When being provided with wherein when utilizing this phenomenon to detect reflectivity detecting unit from the light reflectance of a wall of passage, can also determine whether electrophoresis liquid has reached the part monitoring wall of passage.By equip every end of each passage with the liquid detecting unit of wall light reflectance monitor-type, can make the judgement of the occupied state of electrophoresis liquid.
Judge that by being used to utilize the aforesaid liquid detecting unit thereby the system of the occupied state of electrophoresis liquid forms one with the electrophoresis liquid injection system that utilizes pressure differential and determines that automatically the end sequential of spraying can obtain the robotization of whole electrophoresis liquid spraying.
(be used for fixing system, the baseplate part cooling system of the baseplate part of microchip with sealing lid and be used for the control module of cooling system)
When peeling off and remove from the end face of the baseplate part 103 of microchip when covering sealing 113, behind fixed base board 103, external force is applied to an end that covers sealing 113, is forcing the bonding plane that is displaced between baseplate part 103 and the lid sealing 113 on the vertical substantially direction with an end that will cover sealing 113.Follow this dislocation that covers sealing 113 1 ends, lid sealing 113 has the flexible structure with respect to bonding plane.
In the stage that applies external force, the fixed base board moves to prevent baseplate part 103.Simultaneously, before peeling off and remove the operation of covering sealing 113, cool off the fluid sample of the electrophoretic separation in the channel shaped passage that is present in baseplate part 103, so that whole fluid samples are placed freezing state.This fluid sample is in electrophoretic separation and becomes the alkaline soluble materials dissolving of electrophoresis liquid, the state of formation point.Though the solvent composition of this fluid sample is a water, buffering agent composition and analogous components also are dissolved in wherein, therefore, because solidifying point reduces, so, begin under the temperature of (0 ℃) that is below the freezing point that it is freezing.Therefore, ideally whole fluid samples are cooled fast to the temperature that is starkly lower than freezing beginning temperature,, make the whole fluid samples in the channel shaped passage freezing at once thus to be placed on supercooled state at once.On the other hand, if aqueous solvent slowly freezing under the temperature of freezing beginning temperature, although then the concentration of dissolved substance locate higher because low at the concentration ratio point place of other regional material, so, freezing will the beginning in the place of removing putting.In the case, because the freezing volumetric expansion that causes is the not freezing zone around the compression point, and may cause leak of liquid to go out the channel shaped passage.For fear of this situation, the state of desirable acquisition is, by with fluid sample apace refrigeration to the temperature that is starkly lower than freezing beginning temperature to be placed on supercooled state at once, carry out freezing in the whole inside of channel shaped passage.
Therefore, preferably utilize the baseplate part cooling system, described baseplate part cooling system so that whole passage reaches the consistent temperature that is starkly lower than freezing beginning temperature, obtains supercooled state by the liquid of quick cooling from the bottom surface of the baseplate part 103 of microchip at once.Ideally, refrigerating system can have the structure with the whole bottom uniform contact of baseplate part, and baseplate part fixed system and baseplate part refrigerating system to form the form of one be desirable.
Fixing for baseplate part, although wherein the type that is fixed of the sidewall sections of baseplate part is spendable, the type of the bottom surface of preferred fixed base board.For example, suitably adopt after the plane is processed in the bottom surface of baseplate part bottom surface with baseplate part to be fixed on the type in the precalculated position on the stationary platform of vacuum clip clamping system.Though thickness is originally as several millimeters or littler, but because that the planar dimension of the baseplate part 103 of microchip does not have is little of several millimeters, although but can be not a lot of greatly, but its minor face and long limit also can surpass 10cm, therefore, the surface of preferably using the stationary platform of vacuum clip clamping system wherein is by utilizing chiller (for example, amber ear card (Peltier) device) by the pattern of refrigeration to predetermined temperature.
In addition, forming in the described baseplate part fixed system of one with the baseplate part refrigerating system, when the microchip of stationary platform surface and lid sealing by refrigeration when being starkly lower than the temperature of freezing point (0 ℃), if ambient atmosphere comprises moisture, then it is with condensation and become and freeze.In order to prevent condensation and freeze that the ambient atmosphere of the microchip of stationary platform surface and lid sealing is constituted as in the gaseous state atmosphere that remains on the drying that does not comprise moisture.Particularly, the structure that will have is that the zone itself that comprises baseplate part fixed system and baseplate part refrigerating system is arranged in the airtight sealing container, and the inside of this airtight sealing container remains the atmosphere of dry air and drying nitrogen.
In the state that fluid sample in passage is not frozen, vibration can constitute when making the factor that liquid mixes in passage during carrying, in the step of the above-mentioned electrophoretic separation of operation, the baseplate part 103 of microchip is fixed to the baseplate part fixed system and the baseplate part refrigerating system of described formation one in the fixing position of microchip.The baseplate part fixed system and the baseplate part refrigerating system that form one are provided for electrophoresis equipment, and when the operation that is used for electrophoretic separation had finished, the quick refrigeration of the fixing and baseplate part of the baseplate part 103 of microchip was carried out fast by baseplate part fixed system and the baseplate part refrigerating system that forms one.
In the operational phase that is used for electrophoretic separation, if some other stationary installations are used for baseplate part 103 fixing of microchip, then can utilize the baseplate part fixed system that to realize forming one and baseplate part refrigerating system to be transported to pattern with the tight position contacting in bottom of the baseplate part 103 of microchip.Alternatively, when precalculated position on the equipment is set and be fixed on to the microchip that will cover sealing before electrophoretic separation, even be used for fixing in the case at baseplate part fixed system that will form one and baseplate part refrigerating system, baseplate part fixed system and the baseplate part refrigerating system that also can select to form one can be along with the patterns that operation is carried that is transported into of the microchip that is used for lid sealing to be used.
For with whole fluid samples apace refrigeration to the temperature that is starkly lower than freezing beginning temperature to be placed on supercooled state at once, make the whole fluid samples in the channel shaped passage freezing at once thus, ideally, the refrigeration temperature is set to the scope of be below the freezing point (0 ℃) at least 10 ℃ to 30 ℃, is at least-20 ℃ or lower.When refrigeration during, at once fluid sample is placed supercooled state, thereby make the whole fluid samples in the channel shaped passage carry out freezing at once to described refrigeration temperature.
Comprise by the baseplate part fixed system fixedly microchip baseplate part 103, by the baseplate part refrigerating system via to the fluid sample in the freezing channel shaped passage of the refrigeration of baseplate part 103 and next can carry out automatically by the control module of refrigerating system, and under predetermined condition, realize in order to the temperature controlled serial operation that keeps freezing state.
(being used to peel off and remove the system of covering sealing)
In the present invention, when the baseplate part 103 of the microchip of forming the lid sealing and lid sealing 113 will separate, behind the baseplate part 103 of fixing microchip, the lid sealing 113 that tight adhesion arrives the end face of baseplate part 103 was peeled off and is removed in employing.
Particularly, obtain the bonding strength of adhering state in the predetermined structure that is closely adhered to each other with for the bottom surface of the end face that is released in baseplate part 103 and lid sealing 113, the external force that will have the component on the direction of the end face that is basically perpendicular to baseplate part 103 is applied to the end of covering sealing 113 and covers sealing 113 with bending, and peel off with the form of the end of lift cap sealing 113 upwards with required speed, keep the bending of predetermined curvature simultaneously.In the present invention, when the step of peel-off covers sealing 113, with the channel shaped passage that contacts of bottom surface of lid sealing 113 in remain on the end face of sample of the electrophoretic separation under the freezing state and also peel off fast, therefore, under the sample of freezing electrophoretic separation is stayed state in the channel shaped passage, finish and cover peeling off of sealing 113.
When using according to automatic sample disposal route of the present invention and automatic sample treatment facility, the bonding strength itself of the bottom surface that keeps the state that closely bonds between the bottom surface of the end face of baseplate part 103 and lid sealing 113 to depend on covering sealing 113 and the end face of baseplate part 103.On the other hand, even under refrigeration condition, the bonding strength p of the per unit area between the end face of the sample of the bottom surface of lid sealing 113 and freezing electrophoretic separation 1Also be set at bonding strength p less than the per unit area between the bottom surface of the end face of baseplate part 103 and lid sealing 113 0(p 0＞p 1).
In this process, because the bonding strength between the bottom surface of the end face of baseplate part 103 and lid sealing 113, on the direction of the end face that is basically perpendicular to baseplate part 103, be raised and cover sealing 113 and also do not begin the scope peeled off when crooked even then have an end of lid sealing 113.Under the state of observing than macrobending, the critical value of the beginning of peeling off is arranged.When satisfying the critical value condition, curved shape is limited by δ and L and shows the bowed shape with substantially invariable radius of curvature R, wherein δ be an end place at lid sealing 113 from the displacement of baseplate part 103 end faces, the border that L contacts with each other for the bottom surface from the end face of baseplate part 103 and lid sealing 113 is to the length of the working point that is applied to the external force on the end that covers sealing 113.Therefore, by represent the angle of this arc with θ, satisfy following formula.
L＝R·θ
δ＝R(1-cosθ)
When bending to this shape, be applied to the approximate following expression of power P on the border that contacts with each other, bottom surface of the end face of baseplate part 103 and lid sealing 113, wherein d is a thickness, b is a width, and E is for covering effective Young modulus of sealing 113:
δ＝P·(2L) 3/{4bd 3E}
P＝δ·(4bd 3E)/(2L) 3
When the displacement δ of an end of lid sealing 113 increased to δ → δ+Δ δ, the radius of curvature R of expression curved shape was changed into R → R-Δ R 1, and its arc angle θ becomes θ → θ+Δ θ 1, transition change is expressed as follows:
L＝(R-ΔR 1)·(θ+Δθ 1)
≈R·θ+{R·Δθ 1-ΔR 1·θ}
δ+Δδ＝(R-ΔR 1)·{1-cos(θ+Δθ 1)}
≈(R-ΔR 1)·(1-cosθ+Δθ 1·sinθ)
≈R(1-cosθ)+{R·Δθ 1·sinθ-ΔR 1·(1-cosθ)}
≈R(1-cosθ)+ΔR 1·{θ·sinθ-(1-cosθ)}
When bending to this shape, be applied to the power P+ Δ P on the border that contacts with each other, bottom surface of the end face of baseplate part 103 and lid sealing 113 1Approximate representation is as follows.
P+ΔP 1＝(δ+Δδ)·(4bd 3E)/(2L) 3
Acquisition is slightly peeled off, and for example, reduces P+ Δ P 1→ P.Therefore, it turns back to the state of no longer peeling off.Peeling off when stopping, curved shape shows as the bowed shape with substantially invariable radius of curvature R.
L+ΔL＝R·(θ+Δθ 2)
δ+Δδ＝R·{1-cos(θ+Δθ 2)}
≈R·(1-cosθ+Δθ 2·sinθ)
≈R(1-cosθ)+R·Δθ 2·sinθ
In this stage, be applied to the power P-Δ P on the border that contacts with each other, bottom surface of the end face of baseplate part 103 and lid sealing 113 2Approximate representation is as follows.
P-ΔP 2＝(δ+Δδ)·{4bd 3E}/{2(L+ΔL)} 3
≈(δ+Δδ)·{4bd 3E}/{(2L) 3·(1+3ΔL/L)}
≈(δ+Δδ)·(1-3ΔL/L·{4bd 3E}/(2L) 3
Therefore, work as minimizing: (P+ Δ P 1) → (P-Δ P 2) when taking place, peel off Δ L part.Therefore, the decline of the bonding strength of Δ L part changes corresponding to this: (P+ Δ P 1) → (P-Δ P 2).The bonding strength p of per unit area between the bottom surface of the end face of baseplate part 103 and lid sealing 113 0Use p 0Expression:
(ΔP 1+ΔP 2)＝(3ΔL/L)·(δ+Δδ)·(4bd 3E)/(2L) 3
≈p 0·b·ΔL
In other words, when peeling off when carrying out, estimate 3 (1/L) P ≈ p 0B＞p 1The distortion that surmounts the critical value condition that b is represented (the little bending that its radius of curvature R is very little) need remain on the border that contacts with each other, bottom surface of the end face of baseplate part 103 and lid sealing 113.
Therefore, the external force that is applied on the end of covering sealing 113 is 1/2P, and selects in following scope, wherein keeps
(1/2P)＞p 0·b·L/6
Peel off thereby cause.Certainly, in the case, then simultaneously between the bottom surface of the end face of the sample of freezing electrophoretic separation and lid sealing 113, peel off.
Radius of curvature R on the border (that is the border of, peeling off) that this state need contact with each other in the bottom surface of the end face of baseplate part 103 and lid sealing 113 is less than the radius of curvature R under above-mentioned critical value condition Eq1(R＜R Eq1) state realize down peeling off.
On the other hand, when the crooked radius of curvature R of expression obviously changed, especially when radius of curvature R increases at once and then turn back to initial fractional value, big drawing stress also acted on suddenly on the end face of sample of freezing electrophoretic separation.As a result, although in Frozen Body, there are many flaws (crystal boundary), and because these flaws (crystal boundary) cause platelet exfoliation, so when thin slice was to the bottom surface of covering sealing 113, final sheet adhered to.By keeping the crooked constant state of radius of curvature R of expression, can avoid problem shown in Figure 1, that is, flaw (crystal boundary) peels off and the phenomenon of final sheet adhesion when thin slice is to the bottom surface of covering sealing 113 thus.
The control system of external force application system, lid sealing end induction system and lid sealing end transporting velocity constitutes one, and wherein: the function that described external force application system provides is applied on the end of covering sealing for the external force that will have at the component on the direction of the end face that is basically perpendicular to baseplate part; Described lid sealing end induction system with external force is applied on the end of cover sealing synchronously, the end of sealing is covered in conveying on the direction on the border that contacts between the bottom surface of end face that is basically perpendicular to baseplate part and lid sealing; The function that the control system of described lid sealing end transporting velocity provides is, in the bottom surface that will cover sealing from the step that the end face of baseplate part is peeled off, the end transporting velocity of sealing is covered in control, so that radius of curvature R is remained on predetermined target value, wherein radius of curvature R is represented by the local bending of the borderline lid sealing at the place of peeling off, therefore, for example, can select structure as described below.
(first example embodiment)
The stripping system that is used for lid sealing shown in Figure 5 is for having the type of the roller winding lid sealing of predetermined radii by utilization behind the vacuum draw of the end of covering sealing.Expression cover the radius of curvature R of bending of sealing by the maintenance winding speed constant radius that equals roller that becomes, and also can make the transporting velocity of the end of covering sealing constant.
Cover the desired value of radius of curvature R of the bending of sealing according to expression, regulate the radius of roller, and the setting winding speed.
(second example embodiment)
The stripping system of lid sealing shown in Figure 6 is with the type of its lifting after with the end of clamp clamps lid sealing.In this action, pulling speed cover the desired value selection of radius of curvature R of the bending of sealing according to expression.
(the 3rd example embodiment)
The stripping system of lid sealing shown in Figure 7 is the type of the one or both ends of lift cap sealing.The clamping unit that is used for the one or both ends of the removable cover sealing bottom surface of pushing cover sealing that makes progress.In this action, pulling speed cover the desired value selection of radius of curvature R of the bending of sealing according to expression.
(the 4th example embodiment)
The stripping system of lid sealing shown in Figure 8 is for being clamped the type of back lift cap sealing by vacuum draw unit at an end of lid sealing.In this action, pulling speed cover the desired value selection of radius of curvature R of the bending of sealing according to expression.
The control of pulling speed by utilizing lift arm the anglec of rotation and the vertical moving speed of the pillar of supporting rotating shaft regulate within the required range.
(the 5th example embodiment)
The stripping system of lid sealing shown in Figure 9 is for being clamped the type of back lift cap sealing by vacuum draw unit in the end of lid sealing.In this action, pulling speed cover the desired value selection of radius of curvature R of the bending of sealing according to expression.
In some cases, the platform of fixed base board can reduce, so that with relativeness lift cap sealing.
(the 6th example embodiment)
The stripping system of lid sealing shown in Figure 10 is also for being clamped the type of back lift cap sealing by vacuum draw unit in the end of lid sealing.In this action, pulling speed cover the desired value selection of radius of curvature R of the bending of sealing according to expression.
In some cases, the platform of fixed base board can reduce, so that with relativeness lift cap sealing.
(the 7th example embodiment)
The type of the stripping system of lid sealing shown in Figure 11 is, after the spade pilot unit with pre-determined tilt angle inserts from the end of lid sealing, carries pilot unit, simultaneously along the end of this slant lifting lid sealing.In this action, expression cover the radius of curvature R of bending of sealing by controlling according to the desired value of radius of curvature R selection transporting velocity.
Particularly, the radius of a circle of the inclined-plane of inscribe baseplate part and end face constitutes the radius of curvature R that the bending of sealing is covered in expression.The radius of curvature R that the bending of sealing is covered in expression reduces along with the rising of transporting velocity.Under the situation that transporting velocity is fixed, scalable is by the radius of curvature R of the definite expression bending of this state.
(split system of the lid sealing that is used to separate)
In the present invention, peeling off under the sample of freezing electrophoretic separation is stayed state in the channel shaped passage of lid sealing 113 finished.After this, for example, in above-mentioned second example embodiment, the lid sealing of separation is maintained in the state of clamping clamped unit, the end of covering sealing, and by carrying clamping unit to be removed by the end face from baseplate part.In addition, in the 4th example embodiment to the seven example embodiment,, the lid sealing is removed from the end face of baseplate part by under the state that keeps by the system that is used to peel off, carrying.In the 3rd example embodiment, can the employing pattern be under the state of the one or both ends of lid sealing,, the lid sealing that separates to be removed from the end face of baseplate part by carrying clamping unit respectively by the clamping unit maintenance.
Because the sample of freezing electrophoretic separation remains on the state of staying in the channel shaped passage, so, also can be carried to another equipment when being included in the baseplate part.Be formed at channel shaped passage in the baseplate part under exposed state, and sample can experience and is used for the as above processing of the desivac of example.
(analytical approach that is used for biological sample)
In the analytical approach that is used for biological sample according to the present invention, if isoelectric focusing is as electrophoresis operation, the information on the isoelectric point (pl) and become the polytype protein that can be used for being included in the fluid sample for the treatment of biological analysis then according to molecular wt (M) and quantity (C) that MALDI-MS analyzes.By utilizing this two kinds of information (pl and M), when the difference of indivedual protein tangible molecular weight and isoelectric point (pl) according to it is included in polytype protein for the treatment of in the biological fluid sample of analyzing and experiences so-called bidimensional electrophoresis by making, the form of expression that can sxemiquantitative ground prediction " bidimensional electrophoresis " pattern.Therefore, when by the sample that will collect from patient and the sample of collecting from healthy people compares, and search with search for when relating to " labelled protein " of various diseases the scope that can dwindle sample to be analyzed by " bidimensional electrophoresis " for " the Unidentified protein " that may " labelled protein (marker protein) " be associated of these diseases.
(being used for the chemico-analytic equipment of microchip)
To further specify the preferred entire infrastructure that is used for the chemico-analytic equipment of microchip according to of the present invention below.
Particularly, the chemico-analytic equipment of microchip that is used for according to the present invention is used to handle fluid sample as the electrophoretic separation of target sample, treat that wherein the biological fluid sample of analyzing is formed at the operation that the passage experience of covering in the microchip that seals is used for required electrophoretic separation by utilization, each that makes the multiple material that is included in the fluid sample thus separated on the position, forming point along passage, but this is also can be applied to utilize some other chemical analysis technologies rather than utilize electrophoresis method to separate the time.
In the case, its whole hardware configurations are set at following structure, comprising: the chemical analysis unit 1 of sample that is used for analyzing the passage of microchip; The solution fixed cell 2 that is used for fixing sample for chemical analysis and electrophoresis liquid; And lid sealing separative element 3, lid sealing separative element 3 be used for lid sealing and substrate portion from, with the sample of having fixed in the passage in the baseplate part that exposes microchip.Only otherwise the spare system that then not limiting other individual component or the system of the structures of samples of the equipment of enclosing in can the chemical analysis microchip or being used for described sample application was analyzed in next or ensuing stage is analyzed in influence by chemical analysis unit 1.
Although be not particularly limited, the isoelectric focusing of particularly suitable that the chemical analysis of realizing by the chemical analysis unit among the present invention 1 comprises electrophoretic separation (for example) and allows the sample concentration at indivedual isoelectric points place.In the case, chemical analysis unit 1 can be made up of electrode part and swimming power supply.Voltage supplies to the electrode part by lead by the swimming power supply, and by utilizing electrode part voltage to be applied to electrophoresis liquid in the passage of microchip, to cause occurring electrophoresis.The liquid reservoir cap unit can also further be arranged, with electrophoresis evaporation of liquid in the limiting channel on the lid sealing of microchip.In addition, can also be provided for monitoring the current monitor that applies the levels of current during the voltage.
Chemical analysis unit 1 can also be provided with and be used for liquid reservoir cap unit and/or the electrode part automatic transport induction system to its precalculated position.One or more this backup systems both can have been used separately also and can be used in combination.
Although be not particularly limited, for example, use refrigerating system for the solution fixed cell among the present invention 2, described refrigerating system is used for fixing by described chemical analysis unit 1 chemico-analytic sample or electrophoresis liquid by freezing.
Ideally, the refrigerating system among the present invention can be for passing through the directly type of the baseplate part of contact refrigeration microchip.Can have only a refrigerating system or a plurality of this system, described system can augment the secondary refrigerating system that refrigeration is provided from the lid sealed part side by the liquid reservoir cap unit in the system of baseplate part side.Spendable refrigerating system including, but not limited to, for example, use the refrigerating system of amber ear card device or refrigeratory.
The lid sealing separation vessel 3 that is used for the present invention have by vacuum clamp attract cover sealing and with cover that sealing contacts or the system of fixed cap sealing, pneumatic attraction baseplate part and contact with baseplate part or the system of fixed base board and make fixing lid sealing and baseplate part induction system away from each other.
Although be not particularly limited, by vacuum clamp to attract to be used for the present invention's lid sealing and contact with the lid sealing or the system of fixed cap sealing can for make cover sealing by the vacuum clamping be attracted to fixed system pump unit, will cover sealing and be glued to the gummed unit 12 of fixed system or make and cover sealing contacted or be fixed to fixed system with fixed system lid sealing fixed cell.
Although be not particularly limited, but for example, the system that clamp to attract to be used for baseplate part of the present invention and contact or fixed base board by vacuum can clamp the baseplate part pump unit that is attracted to fixed system, baseplate part is glued to the baseplate part gummed unit of fixed system or makes baseplate part contact or be fixed to the baseplate part fixed cell of fixed system with fixed system by vacuum for making baseplate part.
The lid sealing vacuum draw unit or the baseplate part vacuum draw unit that can be used among the present invention have inlet hole and reduce the pressure letdown system of pressure by inlet hole, and can clamp the object that attracts near inlet hole by vacuum.
Although be not particularly limited, but for example, the induction system that is used for the present invention can be for the chip platform unit of moving substrate portion up and down or lid sealing, to rotate the roller that cover sealing to reel, clamp or hook the clamping unit that covers sealing or baseplate part or hook the unit or make be an opening/closing unit that opens or closes of rotation center, and wherein said induction system makes fixing lid sealing and baseplate part away from each other.
Be used for the chemico-analytic equipment of microchip of the present invention can also be provided with lid sealing-baseplate part connected system by connecting the structure microchip as required, be used for sample and/or electrophoresis liquid spray the passage of microchip into the solution spraying system, be used for separating sample and/or the drying system of electrophoresis liquid and the detecting signal unit that is used to detect chemico-analytic process or result that exposes in the back dry substrate portion will cover sealing and baseplate part.
Although be not particularly limited, but for example, lid sealing-baseplate part the connected system that is used for the present invention can be the positioning and guiding spare such as protuberance, recess, hole or pin that is designed to mate the microchip shape, keeper or the induction system that is used to keep microchip, and described induction system is connected baseplate part and lid sealing with the lid sealing to increase bonding strength by baseplate part being arranged in the precalculated position with the lid sealing and pushing baseplate part.One and a plurality of this systems both can use separately also and can be used in combination.
Although be not particularly limited, for example, the solution spraying system that is used for the present invention can be depressurizing system or pressure application system, and described system produces pressure differential between the opening at the place, two ends of the passage of the microchip that is positioned to produce solution.
Although be not particularly limited, but the drying system that for example, is used for the present invention can be for the heating system that is used to evaporate the freezing sample that is exposed on the baseplate part and/or electrophoresis liquid or is used to distil and is exposed to the freezing sample on the baseplate part and/or the airtight container and the depressurizing system of electrophoresis liquid.By baseplate part being arranged in the airtight container and reducing pressure in the airtight container, freezing sample and/or electrophoresis liquid can distil.Yet, when environment temperature raises, owing to cover freezing sample and/or the electrophoresis liquid that sealing exposes and in liquid, dissolve and spread by removing behind the chemical analysis, so, only can keep under refrigeration condition state by the analysis generation.Yet, by with drying system with described sample drying, sample in the passage and/or electrophoresis liquid can be completely fixed, and irrelevant with environment temperature.
Although be not particularly limited, for example, the detecting signal unit that is used for the present invention can be provided with light irradiation unit.Detecting signal unit has the light detector of the measuring light of being used for wavelength signals (for example, absorbing wavelength or fluorescence) at least.For example, the excitation line irradiation of passage from light irradiation unit, and fluorescence detects by utilizing light detector.This detecting signal unit can used when utilizing chemical analysis unit 1 analytic sample, can use this detecting signal unit by utilizing solution fixed cell 2 to carry out the fixing back of solution after analysis, described detecting signal unit is used for after covering sealing separative element 3 separate cover sealings by utilization according to the passage that keeps exposed vias or by utilizing drying system to keep sample drying and fixing passage to monitor.
Being used for the chemico-analytic equipment of microchip of the present invention can be by using so far one of explanation, a plurality of pattern or it constitutes.
For easy operation, be preferred for the chemico-analytic equipment of microchip of the present invention and also be provided with control module.Control module can be used for the monitoring current level by utilizing the current monitoring unit, with the voltage of control by the power supply supply.In addition, control module can be used for during applying voltage, determines chemico-analytic end according to the levels of current of monitoring, and also is used to control the operation of refrigerating system.In addition, control module can also be used to control the operation that is used for clamping by vacuum the system of attraction, contact or fixed cap sealing, control clamps the operation of the system of attraction, contact or fixed base board by vacuum, thus and the control operation that fixing lid sealing and baseplate part is separated from each other and exposes the induction system of passage.In addition, control module can be used for controlling being used in and covers sealing-baseplate part connected system baseplate part and the induction system that is connected of lid sealing, control being used in the solution spraying system with the depressurizing system or the pressure application system that produce different pressures, controlling the heating system or the depressurizing system that are used in the drying system, and is used for checking the state at the sample analysis of detecting signal unit.
Next, be used for the chemico-analytic equipment of microchip of the present invention with reference to example explanation more specifically.In addition, technical scope of the present invention is not limited to these specific embodiments.
(the 8th example embodiment)
Fig. 3 schematically illustrates graphic as the profile of the equipment that separates according to the execution isoelectric point of the example embodiment that is used for the chemico-analytic equipment of microchip according to the present invention.In this 8th example embodiment, sample separates by isoelectric point and carries out chemical analysis, after sample and electrophoresis liquid are fixed on the state that obtains by the cryofixation analysis, lid sealing and substrate portion from and be removed.As the result who uses airtight container and depressurizing system, be exposed to sample and/or electrophoresis liquid on the baseplate part by distillation, by drying sample is fixed.
Microchip is by the baseplate part 103 with channel architecture and have as the lid sealing 113 of the pore structure of liquid reservoir and form.
At first, baseplate part 103 is installed on the chip platform along the chip guide.The chip platform comprises amber ear card device, inlet hole and induction system.Amber ear card device is also with the cooling system that acts on the refrigeration microchip.Inlet hole is connected to vacuum pump, and baseplate part 103 is fixed clampingly to the chip platform by vacuum.Induction system is covered sealing and baseplate part induction system away from each other with acting on to make.Induction system is also used in lid sealing-baseplate part connected system.
Next, will cover sealing 113 is installed in along the lid guides and covers on the platform.The Gai Taiyu lid guide that is used for this example embodiment forms one, and also plays the effect of covering the sealing fixed system.
After this, the liquid reservoir cap unit is connected to covers on the sealing 113.The liquid reservoir cap unit is provided with electrode part and at the inlet hole of its bottom surface, and electrode partly is disposed in the reservoir part of covering sealing 113.Inlet hole is used for clamping liquid reservoir cap unit and lid sealing 113 by reduce pressure vacuum via inlet hole.The liquid reservoir cap unit is provided with amber ear card device, and described amber ear card device is used when the liquid reservoir cap unit is separated together with lid sealing 113 and acted on the cooling system that covers sealing.In addition, the liquid reservoir cap unit is provided with induction system, described induction system is used and is acted on the induction system that the liquid reservoir cap unit is transported to the precalculated position, plays to be used to make the effect of covering sealing and baseplate part induction system away from each other, and as covering sealing-baseplate part connected system.
Next, chip platform of installation base plate portion 103 utilizes induction system to be moving upward on it, so that being pressed in, baseplate part 103 covers on the sealing 113, thereby by connecting and composing microchip.After this, keep so chip platform position.The liquid reservoir cap unit is carried away from the top of covering sealing 113 to expose the liquid reservoir of microchip.The electrophoresis liquid of sample dissolution is sprayed in the liquid reservoir of lid sealing 113 into.Particularly, in order to carry out isoelectric focusing, the ampholyte with 2% (pharmalyte) is as electrophoresis liquid.When the whole passage of microchip all is filled with electrophoresis liquid, remove the electrophoresis liquid that is retained in the liquid reservoir.Next, negative electrode liquid and anode liquid in liquid reservoir is sprayed at the two ends of passage, and are installed to the liquid reservoir cap unit once more and cover on the sealing 113.
Up to the present the induction system of all references can be operated by utilizing control module.
Voltage is applied to the electrode part by lead from power supply, and the anode and the levels of current between the negative electrode of electrode part are measured by utilizing the current monitoring unit.When levels of current progressively descended along the duration that applies voltage,, then can determine the end that isoelectric point is separated if can measure its levels of current or wattage.
After isoelectric point was separated, operation was used for the cooling system of chip platform and liquid reservoir cap unit, with freezing sample and/or electrophoresis liquid.Next, when clamping the attraction chip via inlet hole by vacuum, the chip platform descends so that lid sealing 113 separates with baseplate part 103.Cover guide and pushed together by the above and below and cover sealing 113 by utilizing, the liquid reservoir cap unit is used as and covers the sealing stationary installation.By the continuous refrigerating base board of the refrigerating system that is used for chip platform portion 103, freezing sample and/or electrophoresis liquid can be exposed on the baseplate part 103.Carve at this moment, baseplate part 103 is positioned at the below of covering sealing 113.Attracting to cover under the state of sealing 113 by the vacuum clamping via inlet hole, the liquid reservoir cap unit is transported to the lid discarding unit top that is positioned in abutting connection with chemical analysis unit 1, and lid sealing 113 is disposed on the bottom surface of covering discarding unit.
After this, the pressure in the airtight container descends by the exhaust opening that is used for whole airtight container emptying.Under the step-down state, finished when distillation when the solution in the passage, stopped pressure and descend, fallen then and turn back to atmospheric pressure.
The drying of being undertaken by cryofixation at the equipment that is used at the sample that chemical analysis isoelectric point on the microchip is separated and fixing, removing aspect the sample and/or electrophoresis liquid, freeze drying example and/or electrophoresis liquid that exposes after covering in the passage, up to the present illustrated processing has proved success.
Industrial usability
Automatic sample processing side according to the microchip of the lid sealing for bioanalysis of the present invention Method and be used for the automatic sample treatment facility of microchip of the lid sealing of bioanalysis can be used for increasing The strong improved reproducibility that is used for the process of sample preparation is utilized by electrophoretic separation through the place The offering sample of reason is further analyzed, and for example, carries out quality analysis and biometric analysis.

Claims (8)

1. one kind is used for treating that the biological fluid sample of analyzing is formed at the microchip of lid sealing by utilization passage carries out required electrophoretic separation operation back, experiences electrophoretic separation and remain on the method that the fluid sample in the described passage is handled automatically, is characterized in that:
The structure that the microchip of described lid sealing has is, the lid sealing that wherein is formed at the channel shaped passage in its baseplate part and seals the end face of described baseplate part obtains to bond to together state with predetermined arrangement, make that the bottom surface of the end face of described baseplate part and described lid sealing is closely bonding each other
After the described passage in the microchip that is formed at described lid sealing by utilization is finished the required electrophoretic separation operation of fluid sample to be analyzed, said method comprising the steps of:
Cooling step, the described baseplate part that has wherein experienced electrophoretic separation and remained on the fluid sample experience microchip by cooling off described lid sealing in the described passage comes the freezing operation that comprises hydrosolvent within it, to obtain to be in freezing point or subfreezing predetermined low-temperature condition;
Peel off the step of described lid sealing, wherein keep being cooled to described predetermined low temperature level by described baseplate part simultaneously the microchip of described lid sealing, the sample that in described passage, keeps having experienced electrophoretic separation under the freezing state that continues,
Peel off and remove the operation of described lid sealing is undertaken by the end that external force is applied to described lid sealing from described baseplate part, so that release has made the tight bonding strength that contacts and obtain adhering state with predetermined arrangement in bottom surface of the end face and the described lid sealing of described baseplate part, thereby peel off the bottom surface of described lid sealing from the end face of described baseplate part, simultaneously with respect to predetermined critical value R Eq1, the radius of curvature R that maintenance is illustrated in the local bending of the borderline described lid sealing of peeling off keeps less than described critical value R Eq1(R＜R Eq1) state; And
The step of separating described lid sealing, wherein after described strip step finishes, in the microchip of described lid sealing, be separated by the described lid sealing that has separated that is adhesively fixed that is released on it with the end face of described baseplate part, then, with the described lid sealing that separates through being used for its carry away from and the operation that keeps so that obtain to make the surface of the sample of the electrophoretic separation under continuing freezing state to be exposed to state in the described channel shaped passage that is formed in the described baseplate part; And
Automatically realize a series of these steps.
2. one kind is used for treating that the biological fluid sample of analyzing is formed at the microchip of lid sealing by utilization passage carries out required electrophoretic separation operation back, experiences electrophoretic separation and remain on the equipment that the fluid sample in the described passage is handled automatically, is characterized in that:
The structure that the microchip of described lid sealing has is, the lid sealing that wherein is formed at the channel shaped passage in its baseplate part and seals the end face of described baseplate part obtains to bond to together state with predetermined arrangement, make that the bottom surface of the end face of described baseplate part and described lid sealing is closely bonding each other
Described equipment comprises the following system that is provided for the microchip of described lid sealing, and the required electrophoretic separation operation of fluid sample wherein to be analyzed is finished by the described passage that utilization is formed in the microchip of described lid sealing:
The system that is used for the described baseplate part of refrigeration, described system are suitable for the arrangement installation that contact with the described baseplate part of the microchip of described lid sealing;
The control module that is used for described refrigerating system, by the system that is used for the described baseplate part of refrigeration of installing with the arrangement that contacts with described baseplate part, described unit can remain on and is under freezing point or the subfreezing predetermined low temperature level state by being cooled to the described baseplate part of major general;
The system that is used for fixing described baseplate part, described system can fix the described baseplate part of the microchip of described lid sealing with the arrangement that contacts with the described system that is used for the described baseplate part of refrigeration;
Be used to apply the system of external force, the function that described system has is, the arrangement of fixing by the described system that is used for fixing described baseplate part with described baseplate part, on the end of described lid sealing, apply the external force of direction, so that release has made the tight bonding strength that contacts and obtain adhering state with predetermined arrangement in bottom surface of the end face and the described lid sealing of described baseplate part with the end face that is basically perpendicular to described baseplate part;
Be used to carry the system of the end of described lid sealing, described system can with the end that external force is applied to described lid sealing by the described system that is used to apply external force on synchronously, on the direction of the Contact Boundary between the bottom surface of end face that is basically perpendicular to described baseplate part and described lid sealing, carry the end of described lid sealing;
Be used to control the system of the speed of the end of carrying described lid sealing, the function that described system has is the transporting velocity of the end of the described lid sealing of control, make by utilizing the process of carrying system that the system and being used to of the end of described lid sealing applies external force that the bottom surface of described lid sealing is peeled off from the end face of described baseplate part, with respect to the critical value R that is scheduled to being used for of on the end of described lid sealing, synchronousing working Eq1, the radius of curvature R of the local bending of the borderline described lid sealing that expression is peeled off remains on radius of curvature R is kept less than described critical value R Eq1(R＜R Eq1) state under;
Be used for the separately system of the described lid sealing of separation, the function that described system has is, behind the EO that described lid sealing is peeled off from the end face of described baseplate part, described system keeps being adhesively fixed with the described lid sealing that separates with the end face of described baseplate part by release, and with of the end face conveying of described lid sealing, so that expose the described channel shaped passage that is formed in the described baseplate part away from described baseplate part; And
Described equipment comprises also and is used to control its automatic operated system that the function that described system has is that the action of each system that realizes the series operation is realized according to the predefined procedure Automatic Program.
3. method that is used for analysis of biological samples, in described method, after the passage in the microchip that is formed at the lid sealing by utilization will treat that the biological fluid sample of analyzing is through required electrophoretic separation operation, the component substances that separates for the point on described passage carries out the quality analysis of the component substances of a separation, these materials will be included in the fluid sample of the electrophoretic separation on the described passage that remains in the microchip that is formed at described lid sealing, it is characterized in that:
The structure that the microchip of described lid sealing has is, the lid sealing of end face that wherein is formed at channel shaped passage in its baseplate part and the described baseplate part of sealing has obtained the state that bonds together with predetermined arrangement, make the end face of described baseplate part and the bottom surface of described lid sealing be closely adhered to each other with
Described method comprises:
Collect step, wherein, after the described passage in the microchip that is formed at described lid sealing by utilization is finished fluid sample to be analyzed required electrophoretic separation operation,
Peel off and remove described lid sealing according to the automatic sample disposal route that is used for the microchip with sealing lid of bioanalysis as claimed in claim 1 by the end face that seals the described baseplate part of tight covering, then,
Collect the sample of electrophoretic separation, the sample of electrophoretic separation remains in the described channel shaped passage that is formed in the described baseplate part to continue freezing state, thereby exposes the surface of the sample of electrophoretic separation;
Be used for freeze-drying and fixing step, wherein continuing sample that freezing state remains on the electrophoretic separation in the described channel shaped passage that is formed in the described baseplate part by freeze-drying, each component substances that separates with each the some place of the point on will the described passage in being formed at described baseplate part is fixed on the relevant point with the form of lyophilized substance;
Be used to provide the step of matrixing agent, the matrixing agent that wherein is used for the MALDI-MS analysis is applied to the described channel shaped passage that is formed at described baseplate part, described matrixing agent is provided to the component substances that is fixed to the electrophoretic separation on the described point with the form of lyophilized substance;
Be used for the step that MALDI-MS analyzes, wherein by utilizing described matrixing agent to carry out the MALDI-MS analysis operation along the described channel shaped passage that is formed in the described baseplate part, molecular weight information with the ionic species that obtains to obtain by component substances through electrophoresis, described component substances through electrophoresis is fixed on the reference point with the form of lyophilized substance, and the positional information of the described point of the molecular weight information of acquisition demonstration ionic species, and
The step that is used for data analysis, wherein show that according to being used to of obtaining the positional information of point of the molecular weight information of ionic species specifies the electrophoresis exponential quantity corresponding to described point, then described information is changed into the concrete electrophoresis exponential quantity of appointment and in the combination of the molecular weight information of the ionic species of measuring along the reference point place of described channel shaped channel location, described electrophoresis exponential quantity and information are inferred from the component substances that is included in the fluid sample to be analyzed.
4. method according to claim 1,
Wherein said electrophoretic separation is operating as isoelectric focusing.
5. method according to claim 2,
Wherein said electrophoretic separation is operating as isoelectric focusing.
6. method according to claim 3,
Wherein said electrophoretic separation is operating as isoelectric focusing.
7. one kind is used for the method that automatic sample is handled, and it is characterized in that:
When finish be used to remove the process of covering sealing after,
According to the automatic sample disposal route of the microchip of the lid sealing that is used for bioanalysis as claimed in claim 1,
Described method also comprises:
Be used for freeze-drying and fixing step, wherein continuing sample that freezing state remains on the electrophoretic separation in the described channel shaped passage that is formed in the described baseplate part by freeze-drying, each component substances that separates with each the some place of the point on will the described passage in being formed at described baseplate part is fixed on the relevant point with the form of lyophilized substance.
8. one kind is used for the equipment that automatic sample is handled, and it is characterized in that:
Except each system of the automatic sample treatment facility of the microchip that is included in the lid sealing that is used for bioanalysis as claimed in claim 2,
Described equipment also comprises:
Be used for freeze-drying and fixing system, utilize this system, the sample that remains on the electrophoretic separation in the described channel shaped passage that is formed in the described baseplate part with the freezing state that continues with following state by freeze-drying, described state is carried to expose the described channel shaped passage that is formed at described baseplate part away from the end face of described baseplate part for the lid sealing that will separate in the system that is used for separating described lid sealing by utilization, makes each component substances of each some place separation of the point on the described passage in being formed at described baseplate part be fixed on the relevant point with the form of lyophilized substance.
CNA2006800116444A 2005-02-10 2006-02-08 Method of automatic sample processing for microchip with sealing lid for bioanalysis and apparatus for automatic sample processing Pending CN101156064A (en)

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JP034821/2005 2005-02-10
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