CN101156072A

CN101156072A - Method of automatic sample processing for microchip with sealing lid for bioanalysis and apparatus for automatic sample processing

Info

Publication number: CN101156072A
Application number: CNA2006800116105A
Authority: CN
Inventors: 藤田真知子
Original assignee: NEC Corp
Current assignee: NEC Corp
Priority date: 2005-02-10
Filing date: 2006-02-10
Publication date: 2008-04-02
Also published as: CN101156064A

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

Having of being used to analyze cover microchip, be used to have the sample treatment that covers microchip, be used to have the automatic sample disposal route of covering microchip, based on the automatic sample treating apparatus of this disposal route and the species analysis device of using this automatic sample disposal route

Technical field

The present invention relates to when be used to analyze cover microchip the time, the disposal route of covering analytic sample in the microchip is being arranged; Automatic processing method; Automatic sample treatment facility based on this method; And the biological substance analytical equipment of using this automatic sample disposal route.The invention still further relates to and use the microchip that covers that this disposal route is carried out separate analysis.More specifically, the present invention relates to be applicable to removing having of lid and cover analytic sample in the microchip and handle the disposal route, automatic processing method of sample, the automatic sample treatment facility that conforms to technology for automatically treating, and relate to the microchip that covers with suitable constructions of being used for described disposal route.

Background technology

For the sample that comprises biological substance or chemical substance, when analysis package is contained in any biological substance that comprises protein and nucleic acid in the sample or any chemical substance so that when discerning them, by comprising that one of electrophoresis and chromatographic various isolation technics come separating bio or chemical substance, then biological assay or chemistry chemical examination are used to show the performance and the quantity thereof of separated material.In these analytical approachs, in the step of separating bio material or chemical substance, according to applied separate mode, for example electrophoresis or chromatography, and use kapillary or column jecket.In the biological assay or chemistry chemical examination of the target substance that is separated, after separating step, in various types of orifice plates (well plate), carry out the measurement of biological respinse, biochemical reaction and chemical reaction.

Especially, little or need under the temperature controlled situation in sample size itself, " microchip " is helpful, and this microchip can produce the passage of low capacity by fine processing, and passage is integrated, and it has compressed needs temperature controlled area.Microchip is combined in the predetermined arrangement of substrate, wherein forms groove shape passage and the channel arrangement with desired plane shape, and cover these passages with lid, substrate and lid bond mutually or are fixing.A kind of method has been proposed, the groove shape channel part that will provide in microchip by this method is used as capillary space or cylindrical space, so that realize separating by electrophoresis or chromatography, and the space, hole that will be provided in the channel part is used for biological assay or chemistry chemical examination (non-patent literature 1:Qinglu Mao etc., Analyst, 124 volumes, 637-641 (1999)).

Multidimensional analysis, promptly each single sample is subjected to a plurality of analyses, is suitable for discerning more accurately biological substance and chemical substance.For example, when protein example has the isopotential point of comprising and two features of molecular wt, can be by obtaining about the information of these two features rather than only rely on a feature to come analysing protein more accurately.Consider this point, a kind of equipment is proposed, the isopotential point that wherein sample is incorporated in the passage in the microchip and is subjected to being equivalent to electrophoresis in passage separates, after this, with MALDI-MS (substance assistant laser desorpted/the MALDI-MS determination method) be applied to along on the sample of channel separation so that collect information (non-patent literature 2:Michelle L.-S.Mok etc. about its position and molecular wt, Analyst, the 129th volume, 109-110 (2004) and patent documentation 1:WO 03/071263A1).Construct this equipment so that " microchip " self cooling and on thermoelectric (al) cooler, be subjected to temperature control, and so that prevent solvent owing to the heating of liquid in the micro-channel is evaporated, its high voltage that applies by following isopotential point to separate causes with the lid sealing at the top of each groove shape passage.

After finishing, with the lid removal and by heated substrates or placement the solvent in the groove shape passage is evaporated rapidly, thereby make its drying and the protein that is separated is solidified in each some position such as the lock out operation of electrophoresis.Suitable host material added in the groove shape passage so that the protein that on microchip, keeps being separated, and carry out along passage that MALDI-MS measures so that each some position is detected.

Patent documentation 1:WO 03/071263 A1t

Non-patent literature 1:Qinglu Mao etc., Analyst, 124 volumes, 637-641 (1999)

Non-patent literature 2:Michelle L.-S.Mok etc., Analyst, the 129th volume, 109-110 (2004)

Disclosure of the Invention

Problem to be solved by this invention

In order to further expand the range of application of lid " microchip ", for being arranged, lid " microchip " needs following function, by after on microchip, finishing lock out operation such as electrophoresis and chromatographic separating measure, lid can separate the operation that the material that separates in the passage is further analyzed then at an easy rate from substrate portion.

Under the situation of carrying out further analysis operation, there is certain situation, promptly need collection of material in next analysis phase or its preproduction phase, this material is separated in advance by the lock out operation that applies various isolation technics in passage.The related problem of step of collecting institute's separate substance in the microchip is following adverse effect easily responsive.

At first, under the situation that adopts following pattern, promptly, a plurality of groove shape passages are formed on the microchip that has a plurality of slypes (electrophoresis path) in the microchip with structure, this structure makes the end face of the groove shape passage that is formed in the substrate tightly be sealed by lid, thereby make the passage that is tightly sealed be suitable for lock out operation such as electrophoresis, this electrophoresis is corresponding to utilizing conventional electrophoretic techniques capillaceous.As the method for this tight seal passage of structure, expectation be by using heat seal or tack coat to make the end face of the substrate portion that wherein forms groove shape passage and the bottom surface of lid part be in firm affixed state.On the other hand, under the situation that reaches more firm affixed state, peel off and remove subsequently the lid part between the bottom surface for the end face that impels substrate portion and lid part, external force is applied to the lid part so that forcibly it is peeled off, but this processing may cause small mechanical vibration.This small mechanical vibration meeting promotes to be present in the liquid mixing in the groove shape passage, so this can be the reason that stimulates the target substance that is separated into narrow point in the groove shape passage to spread once more.And, carrying out along with the carrying out of the diffusion that causes by the concentration gradient in the liquid, will occurring being separated in the groove shape passage diffusion again of the target substance of narrow point in a way in the time that lid partly removes.Therefore, different slype (electrophoresis path) keeps the state of mutual physical separation, thereby can avoid that liquid mixing between the slype (electrophoresis path) and liquid are revealed, the evaporation of solvent and the intrusion of foreign matter, but the diffusion phenomena again of the institute's isolation of target substances that is caused by the special operation of removing the lid part have taken place really.

Especially, under the situation of the material that separates in advance with the form storage of fluid sample, when it being provided for further analysis and maybe needing it is analyzed under the fluid sample state, for example in biological assay or chemistry chemical examination, utilize under the situation of biochemistry in the stationary liquid phase or chemical reaction, need collect the material of the fluid sample state of the former state of separating in advance in the microchip.The fluid sample that liquid reactant need be added to the separate substance in advance of in microchip, collecting and under the situation that they are mixed for reacting, the material that utilizes microchip to separate in advance by lock out operation is collected as the fluid sample that is used for the single part of separating along passage, single part is provided for analyzing then.Collecting separately in the step of these single parts, the problem of existence is, if not quick complete operation will stand following adverse influence.Because the diffusion again in the liquid as time passes, the material that separates with liquid form becomes different states from desired released state.Particularly, unnecessary long-time if this collection operation has consumed, the diffusion again of institute's separate substance will further be carried out, and it can cause significantly departing from of desired released state.

And, if manually be used to peel off and remove the operation of lid part, the time span that is consumed fluctuates with workman's technical merit, and expectation is to realize that high repeatability is purpose and the operation that realizes being used to peeling off and removing the lid part by automatic process.

The present invention will address the above problem, and therefore the object of the present invention is to provide following sample treatment:

By using this method, lid " microchip " is arranged by use use after thus various isolation technics make the lock out operation that sample liquids to be analyzed expected, under the situation that inhibition institute isolation of target substances spreads again, can be used to remove the operation of the lid part that is fixed to the substrate portion end face, this substrate portion is by there being lid " microchip " to constitute, and

Under the situation of the sample that is separated with the oneself state storage of fluid sample, when it is in or is subjected to the analysis of next stage, though this " not expecting phenomenon " such as any change of the diffusion again of the isolation of target substances of liquid condition and initially-separate state can take place, by using this method, by can carry out collecting the operation of institute's isolation of target substances by means of the high repeatability of automatic equipment, suppress this " not expecting phenomenon " simultaneously from " microchip "; And

The objective of the invention is to further to provide based on the automatic sample treatment facility of this automatic sample disposal route and can be used to carry out this sample treatment separately lid " microchip " arranged.

The device of dealing with problems

The inventor has been engaged in profound research and has solved the problems referred to above, and has obtained following a series of discovery.

At first, they notice two phenomenons of " target substance spreads again ", they are liquid by by peeling off that the milli machine vibration that causes with the special action of removing the lid part mixes and spread by the concentration that the concentration gradient of the target substance that is separated causes, even these two phenomenons are related with this situation that will material after finishing such as the lock out operation of electrophoresis remains on solution in the passage that is formed in the lid " microchip ".Thus, the inventor finds to be set to wherein to be difficult to take place in the solid state shape of material internal migration and replaces and keep solution state, will significantly prevent two phenomenons of " target substance spreads again ".Particularly, have been found that, after the lock out operation of finishing such as electrophoresis, cool off the operation that remains on the solution in the passage fast, so that make the hydrosolvent that comprises wherein freezing, keeping this freezing state to peel off and remove the operation of lid part simultaneously then, the concentration diffusion that can avoid the liquid mixing that causes by small mechanical vibration and concentration gradient to cause thus by the target substance that is separated.

In addition, the inventor finds, in lid " microchip " is arranged, the cross sectional shape that wherein is formed at the groove shape passage in the substrate portion rectangle that to be the top equate than the trapezoidal or top of bottom side length and base, and with the cohesive strength p of basal surface sample segment that contact, frozen state (top) the per unit passage length of lid part _TopThe cohesive strength p that has surpassed sample segment (following and two sides) the per unit passage length that contacts with the side wall surface of substrate portion _Bottom, the sample of frozen state leaves the side wall surface of substrate portion and changes into the state that is bonded to lid part basal surface.The inventor finds, by utilizing this situation, freezing sample can be taken out from " microchip " with the state that is bonded to removed lid basal surface partly.More specifically, at first,, there is lid " microchip " to remain under the cryogenic conditions under the freezing point with whole in order in passage, to place the sample that electrophoresis or other modes are separated with the freezing state that continues.Even near a large amount of rapidly at low temperatures their cohesive strengths of forfeiture of the lid-substrate contacts face that room temperature or room temperature, has enough cohesive strengths, sometimes even fall to below the shown cohesive strength of frozen state sample.In this case, when peeling off at the interface between lid-substrate contacts face, between the basal surface of the sample of frozen state and lid part, can not peel off.The inventor also finds, when with the cohesive strength p of basal surface sample segment that contact, frozen state (top) the per unit passage length of lid part _TopThe cohesive strength p that has surpassed sample segment (following and two sides) the per unit passage length that contacts with the side wall surface of substrate portion _BottomThe time, the frozen state sample leaves the side wall surface of substrate portion and changes into the state that is bonded to lid part basal surface.Also find, replacement wherein forms the end face and the lid basal surface partly of the substrate portion of groove shape passage by using heat seal or tack coat with more firm bond state setting, the end face and the lid basal surface partly that are applied to the substrate portion that wherein forms groove shape passage by the external pressure with machinery keep sealing property, so that only strengthen faint bonding, rather than similar situation when cancelling, the external pressure of this machinery takes place.

The inventor finds that also because freezing sample solution has the advantage and the therefore easier processing of solid, therefore freezing sample solution can be divided into single part, and each part can be transferred to preposition so that further handle.

Based on above discovery, the inventor has finished the present invention by checking:

Under the situation that the cross sectional shape of the groove shape passage in the substrate portion that is formed at lid " microchip " is the top than the trapezoidal or top of the following length of side and the following rectangle that equates, with the cohesive strength p of lid basal surface sample segment that contact, frozen state (top) per unit passage length partly _TopThe cohesive strength p that has surpassed sample segment (following and two sides) the per unit passage length that contacts with the side wall surface of substrate portion _Bottom,

Can operate, so that remove bonding and be fixed to and have the lid part that having of sample covered the substrate portion top surface in the microchip, this sample is separated in the groove shape passage in being formed at substrate portion, keeps being bonded on the basal surface of lid part with freezing substantially state

By making the separating liquid that remains in the passage be subjected to the operation that makes contained solution freezing,

After the lock out operation of expecting, cover fluid sample that the passage in the microchip analyzes and apply separation method being formed at such as electrophoresis by utilization, and

Peel off and remove the operation of lid part, this lid partly seals the top surface of the groove shape passage that is formed in the substrate portion,

When remaining on the sample that separates in the passage with freezing substantially state;

Comprise that also the sample with frozen state is divided into single part, it is transferred to the precalculated position and further handles; And

These a series of operations can be automatic.

Thus, the microchip that covers according to the present invention is:

This microchip is characterised in that:

Being formed at this cross sectional shape that groove shape passage in lid " microchip " substrate portion is arranged is the top rectangle more equal than the trapezoidal or bottom and upper segment of the following length of side; And this microchip comprises lid and the substrate that is made of this material:

Cohesive strength p with the per unit passage length of the part (top) at basal surface (top) sample that contact, frozen state the place of lid part _TopThe cohesive strength p of per unit passage length that has surpassed the part (following and two sides) at the sample place that contacts with the side wall surface of substrate portion _Bottom

In addition, treatment in accordance with the present invention has the method for covering the sample in the microchip to be:

Fluid sample that will be analyzed is being formed at by utilization after the passage that covers in the microchip applies the lock out operation that predetermined isolation technics expects, this method is used for handling and remains on the sample of separating liquid that is formed in the passage that covers microchip, is characterised in that:

The described microchip that covers has following structure, the groove shape passage that wherein is formed in its substrate portion has reached the state that bonds together with predetermined arrangement with the lid part that seals this substrate portion top surface, so that make the top surface of substrate portion and lid basal surface partly bonded to one another securely

Be formed at the passage that covers in the microchip by utilization to after analyzed fluid sample is finished the lock out operation of expectation, this method may further comprise the steps:

Cooling step wherein makes the sample of separating liquid that remains in the passage be subjected to refrigeration operation, and this refrigeration operation describedly has the substrate portion of covering microchip to make the hydrosolvent that comprises wherein freezing so that realize freezing point or predetermined low temperature level condition below freezing by cooling off;

The step of peel-off hd part, its purpose is the substrate portion of covering microchip is arranged under predetermined low temperature by keeping described, makes sample leave groove shape passage with the state that is bonded to lid part basal surface, simultaneously the sample that is separated is remained on and continue under the freezing state

By external force being applied to lid end partly, carry out peeling off and removing the operation of lid part, so that keeping and predetermined threshold R from substrate portion so that the basal surface of lid part and the top surface of substrate portion are peeled off _Eq2In the time of relevant condition, discharge the operation of cohesive strength, this cohesive strength closely contacts the basal surface of the top surface of substrate portion and lid part each other and reaches bond state with predetermined arrangement, and this condition is meant that the radius of curvature R that is demonstrated by the interface upper cover part local bending at the place of peeling off is greater than described threshold value R _Eq2(R＞R _Eq2); And

The step of separate cover subdivision, wherein, after described strip step finishes, transmit as follows and turning operation, to partly transmit top surface by discharging with the bonding of the top surface of substrate portion and the fixing lid that separates away from substrate portion, keep a kind of state simultaneously, the state that described sample separation is in freezing state substantially and keeps boning with lid basal surface partly under this state, top surface and basal surface with the lid part is turned upside-down then, the cover section branch that is separated remained on make the separated sample of the basal surface that is bonded to the lid part that is in basic freezing state be exposed at its lip-deep layout;

Wherein this series of steps is carried out in turn.It also can be the automatic sample disposal route, and wherein this series of steps is carried out automatically.In the method, be formed at the passage that covers in the microchip by utilization the lock out operation that analyzed fluid sample is applied is comprised electrophoresis and liquid phase chromatography, for example, electrophoresis, particularly isoelectronic focusing are suitable.

And treatment in accordance with the present invention has the method for covering the sample in the microchip also can have following formation:

After the described step that is used to dismantle the lid part is finished,

This method also comprises:

Division step, wherein keeping by when applying predetermined isolation technics sample that separate, that remain on basic freezing state and be bonded to the state of lid part basal surface, by the groove shape channel separation of sample from be formed at substrate portion collected this sample, then the sample that is separated is segmented into a plurality of fragments along described passage

The component substances that is separated into mottled (spotpoint) in the groove shape passage in being formed at substrate portion is included in any of described a plurality of fragments; And

Handle the step that fragment is dissolved again, wherein be included in any of the corresponding a plurality of fragments of a part of described sample separation in, each sample fragment of being in freezing state is subjected to dissolution process more respectively, so that prepare the sample liquids of each fragmentation.This series of steps also can be carried out automatically.

And, be used to have the automatic sample treatment facility that covers microchip to be according to of the present invention:

With analyzed fluid sample by apply utilize the lock out operation that the predetermined isolation technics be formed at the passage that covers in the microchip expected after, the fluid sample that is separated remains on and is formed in the passage that covers in the microchip, this apparatus characteristic that is used for handling automatically is

The described microchip that covers has following structure, the lid part that wherein is formed at groove shape passage in its substrate portion and hermetic sealing substrate part top surface reaches the state that bonds together with predetermined arrangement, so that the basal surface of the top surface of substrate portion and lid part closely bonds each other

This equipment comprises the following system that is provided in to cover microchip, has to cover at this to be formed at this by utilization in microchip and to have the passage that covers in the microchip to finish the lock out operation that analyzed fluid sample is desired:

The system that is used for the cooling base part, this system are suitable for and the described device that has the substrate portion of covering microchip to contact and arrange;

The control module that is used for cooling system, this unit can cool off by the substrate portion cooling system is installed with the layout of contact substrate part, thereby remains on freezing point or predetermined low temperature level condition below freezing to major general's substrate portion;

The system that is used for fixing substrate portion, this system can be with the fixing described substrate portion of covering microchip that has of the layout that contacts described substrate portion cooling system;

Be used to apply the system of external force, this system has the function that the end of lid part is applied external force, this external force has the component on the direction that is basically perpendicular to the substrate portion top surface, closely contacts the cohesive strength that also reaches the bond state of predetermined arrangement thus so that discharge the top surface that makes substrate portion each other with the basal surface of lid part;

Be used to transmit the system of lid part end, this system can be in the end that external force is applied to the lid part by described external force application system, the end that the transmission cover section is divided on the direction of the contact interface between the basal surface of top surface that is basically perpendicular to substrate portion and lid part;

The system that is used for the speed of control transmission lid part end, this system has the function of the transmission speed of control lid part end, so that by use external force application system and lid part end-transfer system with the basal surface of lid part from the technology that the top surface of substrate portion is peeled off, this system works to the end of described lid part simultaneously, the radius of curvature R that is demonstrated by the interface upper cover part local bending at the place of peeling off remains under a kind of condition, itself and predetermined threshold value R _Eq2Relevant, promptly radius of curvature R is greater than described threshold value R _Eq2(R＞R _Eq2);

Be used to separate the system of separated lid part, this system has following function, with lid part after the EO that the top surface of substrate portion is peeled off, this system keeps by discharging the fixing and lid part that separate from the substrate portion top surface of bonding, with the top surface of its transmission away from substrate portion, top surface and basal surface with the lid part is turned upside-down then, so that the basal surface of lid part is upwards exposed; And

This equipment further comprises and is used to control its automatic operated system, and this system has the function that the action that makes each system that finishes sequence of operations is finished automatically according to predetermined process.In this case, be formed at the passage that covers in the microchip by use the lock out operation that analyzed fluid sample is applied is comprised electrophoresis and liquid phase chromatography, for example, electrophoresis, particularly isoelectronic focusing are suitable.

And, be used to have the automatic sample treatment facility that covers microchip also can have following structure according to of the present invention:

Except that above-mentioned each system,

This equipment also comprises:

The system that is used for segmentation, this system has the function that the sample that will be in basic frozen state cuts into fragment, wherein, for by applying the sample that predetermined isolation technics is separated, this sample keeps freezing substantially state, by sample and the groove shape channel separation that is formed in the essential part are collected sample, keep it to be in the state that is bonded to lid part basal surface simultaneously, the sample that has separated is segmented into a plurality of fragments that belong to described passage, so that prepare to be in a plurality of sample fragments of substrate frozen state, and

Be used to handle the system that fragment is dissolved again, this system has distributed function and the molten again function of heat, wherein will be distributed to each well of many wells sample panel, and make each sample fragment be subjected to again dissolution process then to prepare the liquid of each fragment sample by each that utilize a plurality of sample fragments that are in basic frozen state that the described sample that system segment separated that is used for segmentation prepares.

The present invention also provides a kind of sample analysis method, wherein, and after the lock out operation that has the isolation technics such as electrophoresis of covering microchip to carry out by utilization is finished,

By the microchip that covers with said structure according to the present invention is applied the automatic sample disposal route,

In the step of the lid part of peeling off and remove hermetic sealing substrate part top surface, by with separate in the groove shape passage of sample from be formed at substrate portion collect remain on basic frozen state, by applying the sample that separates such as the isolation technics of electrophoresis, hold it in the state that is bonded to lid part basal surface simultaneously;

After the step of the lid part of peeling off and remove hermetic sealing substrate part top surface is carried out, this peels off and removes step after by means of the lock out operation such as electrophoresis, with remain on basic frozen state, be segmented into a plurality of fragments by applying the sample that separates such as the isolation technics of electrophoresis along described passage; Then

Each fragment is carried out analysis operation, biological example chemical examination or chemistry chemical examination.

Therefore, under the situation of selection electrophoresis, be according to sample analysis method of the present invention as isolation technics:

The method that is used for analysis of biological samples, wherein, be formed at by utilization the passage that covers in the microchip make want operation that analyzed fluid sample expected after, outside remaining on of electrophoretic separation is formed at fluid sample in the passage that covers in the microchip, the component substances point that separates on the described passage is divided into a plurality of fragments along passage, carry out the biological assay or the chemistry chemical examination of the some separated component material that comprised in the fragment then, be characterised in that:

The described microchip that covers has following structure, wherein the partly fixed surperficial lid part of groove shape passage that forms in its substrate portion and hermetic sealing substrate reaches the state that bonds together with predetermined arrangement, so that the basal surface of the top surface of substrate portion and lid part closely bonds each other

This method comprises:

Collect step, wherein, be formed at by utilization after the passage that cover in the microchip operates the electrophoretic separation of wanting analyzed fluid sample to finish expectation,

Have the automatic sample disposal route that having of said structure covered microchip according to being used to, will partly peel off and remove away by the lid that seals tight covered substrate part top surface, then

By sample is isolated it from the groove shape passage that is formed at substrate portion, thereby collect the electrophoretic separation sample that remains on basic freezing state when keeping adhering to the state of lid part basal surface;

The step that is used for segmentation, the electrophoretic separation sample that wherein will remain on basic freezing state is segmented into a plurality of fragments along described passage, and

Make the component substances that is divided into point-like that is arranged in the groove shape passage that is formed at substrate portion be included in any of described a plurality of fragments;

Be used to handle the step that fragment is dissolved again, wherein make to be included in to be subjected to dissolution process more respectively, to prepare the sample liquids of each fragment corresponding to each sample fragment in any of a plurality of fragments of a described electrophoretic separation sample part, frozen state;

The step of each sheet segment limit is described,,, carries out identification with the corresponding electrophoresis exponential quantity in fragment two ends based on the positional information at fragment two ends on the passage for along being formed at each of a plurality of fragments that the groove shape passage in the substrate portion cuts apart;

Whether the step that is used for the assay fragment wherein makes the sample liquids of each fragmentation be subjected to biological assay and is contained in the fragment with any component substances of determining to demonstrate special characteristics that can be by assay identification; And

Be used to analyze the step of data, wherein determine based on the biological assay analysis result of fragment whether the component substances that demonstrates the special characteristics of discerning by described biological assay analysis has been divided into point-like in the sheet segment limit of just studying, and

About the information of the electrophoresis exponential quantity scope of each fragment identification range, wherein this information is determined to have obtained component substances along groove shape passage, and this component substances demonstrates can be by the special characteristics of described assay identification.

The invention effect

By utilizing according to the microchip that covers of the present invention, be used to have the sample treatment that covers microchip, be used to have the automatic sample disposal route of covering microchip and be used to have the automatic sample treatment facility that covers microchip, lid " microchip " is being arranged to wanting after analyzed sample liquids carries out lock out operation such as electrophoresis by utilization, when the target substance that suppresses to be separated spreads again, can carry out with adhere to and be fixed on the substrate portion top surface cover arranged peel off and remove operation, this substrate portion and have cover to constitute lid " microchip " wherein, and can carry out automatically with repeatability highly.

In addition, after the automatic operation with high repeatability of peeling off and removing the lid part, utilizing by applying in the specimen preparation operation of the sample that separates such as the isolation technics of electrophoresis before further analyzing, above all stages and the analysis of fluid sample, biological example chemical examination or chemical assay, can suppress to spread again or the change of released state, and the automatic operation that can realize having the height repeatability.Therefore, even want analyzed sample liquids to be subjected to lock out operation in a large number such as electrophoresis, the technology that is used to handle sample also can reach the repeatability of height, wherein makes the sample that lock out operation separated by applying such as electrophoresis be subjected to sample preparation for being equipped with sample of further analysis is provided.

Accompanying drawing is briefly described

Fig. 1 is the diagram that has schematically illustrated problem to be solved by this invention;

Fig. 2 is the diagram that has schematically illustrated the microchip channel example of using among the present invention;

Fig. 3 has schematically illustrated the diagram of using among the present invention of covering the microchip structure example that has;

Fig. 4 has schematically illustrated the diagram of using among the present invention of covering another topology example of microchip that has;

Fig. 5 has schematically illustrated the diagram that can be used for according to the example of the lid part stripping system in the automatic sample treatment facility of the present invention, and the principle of work that stripping system utilized in first exemplary embodiment has been described;

Fig. 6 is the diagram that has schematically illustrated the example of the lid part stripping system that can be used in the automatic sample treatment facility of the present invention, and the principle of work that stripping system utilized in second exemplary embodiment has been described;

Fig. 7 is the diagram that has schematically illustrated the example of the lid part stripping system that can be used in the automatic sample treatment facility of the present invention, and the principle of work that stripping system utilized in the 3rd exemplary embodiment has been described;

Fig. 8 is the diagram that has schematically illustrated the example of the lid part stripping system that can be used in the automatic sample treatment facility of the present invention, and the principle of work that stripping system utilized in the 4th exemplary embodiment has been described;

Fig. 9 is the diagram that has schematically illustrated the example of the lid part stripping system that can be used in the automatic sample treatment facility of the present invention, and the principle of work that stripping system utilized in the 5th exemplary embodiment has been described;

Figure 10 is the diagram that has schematically illustrated the example of the lid part stripping system that can be used in the automatic sample treatment facility of the present invention, and the principle of work that stripping system utilized in the 6th exemplary embodiment has been described;

Figure 11 is the diagram that has schematically illustrated the example of the lid part stripping system that can be used in the automatic sample treatment facility of the present invention, and the principle of work that stripping system utilized in the 7th exemplary embodiment has been described;

Figure 12 is the diagram that has schematically illustrated another example of passage of the microchip that uses among the present invention.

The following symbol that uses among the figure has following listed implication respectively:

There is cover on 101 planes

102 binding resin retes

103 substrate portion

105a, 105b, 105c, 105d liquid reservoir

The 107a injection channel

The 107b split tunnel

110 electrode tip fixed parts

112 cover microchip

113 lid parts

Carry out optimal mode of the present invention

To explain the present invention in detail below

Pending sample is the fluid sample that has separated in sample treatment that uses in according to the present invention or the automatic sample disposal route, this fluid sample is formed at the passage that covers in the microchip and prepares wanting analyzed fluid sample to separate by utilization, thus by applying a plurality of materials that on the position, comprise in the separating liquid sample such as the isolation technics of electrophoresis, so that form point along channel location.

When predetermined lock out operation finished, the separating liquid sample remained on liquid state and is formed in the passage that cover in the microchip, this separating liquid sample stood lock out operation by the isolation technics of utilization such as electrophoresis.When having stood by utilizing such as the sample of separating liquid of the lock out operation of the isolation technics of electrophoresis as with the sample of post analysis the time, it must be subjected to according to the specimen preparation operation with after-applied analytical technology.

For example, analytical technology subsequently is to utilize under the biological assay or chemical situation about chemically examining of the reaction in the liquid phase, carry out sample is divided into the operation of a plurality of fragment samples, on the position, separately be included in any of a plurality of fragments of separating along passage with the every kind of material that forms point so that make along passage.After this, analyze each fragment sample according to predetermined reaction product by chemical examination, whether any target substance that is comprised in reacting thus is present in the evaluation in the fragment sample, if exist, comprises its how much quantity in the fragment sample.The form that is used to handle according to microchip of the present invention, sample treatment, automatic sample disposal route and automatic sample treatment facility, wherein, carrying out by after utilizing lock out operation such as the isolation technics of electrophoresis, the sample separation that is formed in the passage that covers in the microchip is divided into a plurality of fragment samples along passage, and does not weaken the released state between the material that separates on the position.

(by utilize separate such as the technology of electrophoresis, want processed fluid sample)

At first, will describe the fluid sample that separates such as the isolation technics of electrophoresis by utilizing below, its sample treatment, automatic sample disposal route or automatic sample treatment facility that will be used according to microchip of the present invention is handled.

Be formed under the situation of the passage in the microchip of lid sealing in utilization, can apply the electrophoretic separation that is equivalent to conventional Capillary Electrophoresis.Particularly, in the biomolecule that is contained in the fluid sample that will analyze is under the situation of protein, can use equipotential to focus on or phoretic (phoretic) separation, this equipotential focuses on the difference of separating by utilizing the isopotential point that is demonstrated by each protein and separates different protein, and this phoretic separation comes from the difference that causes electric conductivity speed between the atom of molecular wt difference by utilization separated from one another with protein.In being contained in fluid sample, to want analyzed biomolecule be under the situation of nucleic acid molecules, can use phoretic separation, it separates different nucleic acid molecules by the difference of utilizing matrix length (length of the base), promptly utilizes the difference that causes electric conductivity speed between the atom that is caused by molecular wt.

In these cases, suitably select to be formed at the flat shape of the passage self that covers in the microchip, the layout and the passage length of passage according to employed electrophoresis separating method.For example, can select to have the channel architecture of flat shape shown in Figure 2.In channel architecture shown in Figure 2, on the end face of substrate portion 103, be provided for equipotential and focus on split tunnel 107b and the injection channel 107a that separates, this injection channel is used to introduce the biomolecule that will be focused on the path 10 7b.At the two ends of split tunnel 107b, form liquid reservoir 105d and 105c, and will be used for providing the acidity of pH gradient and akaline liquid to be injected into liquid reservoir 105d and 105c, be used for providing betwixt the electrical terminal of electric field to be inserted into liquid reservoir.Liquid reservoir 105a and 105b also are formed on the place, two ends of injection channel 107a.Be used for providing the electrode of electric field also to insert liquid reservoir 105a and 105b, be used for the electric field that protein moves so that in the 107a of injection channel, produce.At the two ends of split tunnel 107b, form liquid reservoir 105d and 105c, and will be used for providing the acidity of pH gradient and akaline liquid to be injected into liquid reservoir 105d and 105c, be used for providing betwixt the electrical terminal of electric field to be inserted into liquid reservoir.Liquid reservoir 105a and 105b also are formed on the place, two ends of injection channel 107a.Be used for providing the electrode of electric field also to insert liquid reservoir 105a and 105b, be used for the electric field that protein moves so that in the 107a of injection channel, produce.

In employed electrophoretic separation is under the situation of equipotential focusing, can also select to dispense the channel architecture of the injection channel 107a in the channel architecture shown in Figure 2, only is provided for equipotential and focuses on the split tunnel 107b that separates.Figure 12 shows only to provide and is used for the channel architecture example that equipotential focuses on the split tunnel 107b that separates.

Liquid reservoir

105d and 105c are formed on the two ends that are implemented in the split tunnel 107b in substrate portion 103 end faces, and the acidity and the akaline liquid that will be used for producing the pH gradient are injected into these liquid reservoirs 105d and 105c.Insertion is used for applying the electrode terminal of electric field so that be used for the electric field that protein moves in split tunnel 107b generation.Incidentally, though split tunnel 107b shown in Figure 12 be shaped as the single track structure, it can expand to multiple tracks type microchip, and a plurality of groove shape passages wherein are provided in the end face of substrate portion 103.

Except that aforementioned electrophoretic separation, be formed at by utilization and cover other isolation technics that the passage in the microchip uses and comprise that liquid phase chromatography separates.Being constructed as follows of this technology is formed at the column filler that the passage that covers in the microchip is used for liquid phase chromatography and fills, and makes moisture eluant, eluent medium flow to the other end from an end of passage at a predetermined velocity.Be suitable for filling and be formed at the column filler that covers the passage in the microchip and comprise those of absorption sectional area with subparticle size and relative expansion.The column filler example that is suitable for having the column passage of this fine sectional area comprises silica granule and polymer beads.The length L that is formed at the column passage that covers in the microchip is chosen in 10mm to the 2000mm scope, preferably in 50mm to 400m scope.What expect is that the pore size of employed column media is chosen in 1nm to the 50nm scope, and the particular table area of column filler is chosen in 30mm ²/ g to 800mm ²In/g the scope.

(the electrophoresis operation that the structure of covering microchip is arranged and use it)

Covering microchip is made of substrate portion 103 and lid part 113, in the top surface of substrate portion 103, form groove shape passage, this groove shape passage has the cross section that is configured as the rectangle that trapezoidal or top margin that top margin is longer than the base and base equate, the top surface by these lid part 113 groove shape passages is tightly sealed covering.Incidentally, the hole that is used for the liquid injection is formed at lid part 113, and it matches with the liquid reservoir that groove shape channel end place provides respectively, and the end face of groove shape passage is covered fully by it simultaneously.The binding resin rete 102 that lid part 113 is bonded in substrate portion 103 top surfaces by being used on plane cover base section 101 with maintenance lid part 113 physical strength functions and the lid base section bottom surface constitutes.Be used for hole that liquid injects and liquid reservoir 105d and the 105c and liquid reservoir 105a and 105b that are based upon in plane cover base section 101 and the binding resin rete 102 aim at.And also use when the electrode terminal that will be used to apply electric field is inserted into liquid reservoir 105d and 105c and liquid reservoir 105a and 105b in the hole that liquid injects of being used for that is based upon in plane cover base section 101 and the binding resin rete 102.Incidentally, in some cases, plane cover base section 101 can be constituted and the binding resin rete 102 of its bottom surface that is used to bond with top surface identical materials with substrate portion 103.When same material being used for these two parts, can make them with integrated form in advance.

Add and be fixed for applying the electrode terminal of electric field for the hole that is used for the liquid injection in plane cover base section 101, electrode terminal fixed part 110 was pre-assembled to plane cover base section 101. before the electrophoresis operation, the electrode terminal that is used to apply electric field can fix by utilizing electrode terminal fixed part 110, and in the transmit stage of handling from the electrophoresis EO to automatic sample, the electrode terminal that will be used to apply electric field is removed from electrode terminal fixed part 110.This lid base section 101 and electrode terminal fixed part 110 can be constituted and assembled or be made of same material by different materials, and under latter event, they can be in advance with integrated form manufacturing.After the microchip fixed system by electrophoresis equipment will cover the microchip layout and be fixed on the precalculated position, by use electrode terminal additional/that the removal system can finish these of the electrode terminal that is used to apply the used electric field of electrophoresis operation is additional and remove operation, for electrode terminal additional/ pre-determined the mutual alignment of a plurality of electrode terminals that are used to apply the electric field that will use the removal system.Certainly can also be by manual operation additional and remove electrode terminal and be fixed with and cover microchip, but can make the microchip fixed system that offers electrophoresis equipment and electrode terminal additional/the removal system constructs with automatic operation format.Though special expectation is in the present invention, after finishing the electrophoresis operation, automatically carry out a series of sample preparation operations by remaining on its locational microchip self, but preferably have the automatic operation format of additional and removal electrode terminal and be fixed with the operation of covering microchip.

Incidentally, in exemplary configurations shown in Figure 3, be based upon the form assembling and the fixed electorde terminal fixed part 110 of the sidewall sections in the hole that is used for the liquid injection in the plane cover base section 110 with structure, can also select following structure, wherein to be coupled to the form assembling and the fixed electorde terminal fixed part 110 of the upper end that is based upon the hole that is used for the liquid injection in the plane cover base section 101, another kind of structure as shown in Figure 4.

Substrate portion 103 and lid part 113 are aimed at so that construct a kind of structure mutually according to each hole that is used for the liquid injection and the position of liquid reservoir, wherein by adhering to each other by means of the bottom surface of the end face of substrate portion 103 and lid part 113, promptly, closely cover groove shape path 10 7a with lid part 113 by means of binding resin rete 102.For the combination of plane cover base section 101 and binding resin rete 102, adopt the combination that demonstrates high-combination performance; And, select a kind of form that it is peeled off on the end face and the bonding plane between the binding resin rete 102 that occurs in substrate portion 103 when when lid part 113 is peeled off and removed to the step of back.Thus, bonding plane between the end face of substrate portion 103 and the binding resin rete 102 will present enough cohesive strengths to realize enough coherent condition closely, this enough coherent conditions closely are not filled in the problem that the groove shape path 10 7a of protein liquid from be formed on substrate portion 103 end faces in the passage reveals or leach, 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 or automatic sample treatment facility, preferably, the adhesion strength of binding resin rete 102 self is set to low, but keep the top surface of substrate portion 103 and the tight adhesion state between the binding resin rete 102 by means of loading enough external force, to solve deficiency.Keep the device of substrate portion 103 and lid part 113 tight adhesion as loading external force, can use the load-carrying application system that on the upper surface of lid part 113, applies load-carrying.What expect is the form of selecting to be used for this load-carrying application system, and wherein load-carrying can be evenly dispersed on the whole adhesive surface of substrate portion 103 and lid part 113 substantially.In addition, preferably, use a kind of form of operation automatically that allows, add/the removal system, remove the load-carrying in the operation of peeling off and removing lid part 113 as microchip fixed system and electrode terminal.For example, load-carrying application system and electrode terminal additional/the removal system can be integrated in case make electrode terminal additional/the removal system is suitable for electrode terminal after applying load-carrying by the load-carrying application system.

For the end face of substrate portion 103, select this material, this material can be realized the processing degree of accuracy of expecting so that make fine groove shape path 10 7 therein when carrying out the processing of aforementioned microtexture.According to employed electrophoresis method, the cross sectional shape of groove shape passage is chosen in the channel width (W of 5 μ m to 1000 μ m ₁) and channel depth (D ₁) in the scope.The fine groove shape passage of this " microchip " is mainly used in the electrophoretic separation operation of using indivisible fluid sample, replaces Capillary Electrophoresis.Therefore, that expectation is the sectional area (D that selects fine groove shape passage ₁* W ₁) make it the same little with interior sectional area capillaceous, for example in the sectional area scope that is no more than 100 μ m interior diameters.On the other hand, by the material of substrate portion 103 with by definite processing accuracy, suitably the selector channel degree of depth (D by means of the fine processing of calculating groove shape passage ₁)/channel width (W ₁) ratio (D ₁/ W ₁).Usually, because too high ratio (D ₁/ W ₁) will increase the difficulty of handling, therefore expectation is that ratio is chosen in 1/100≤D ₁/ W ₁In≤10 scopes.

On the other hand, in the present embodiment, replace electrophoresis method, use chromatography to carry out lock out operation, select this pattern with column agent filling channel.With in the pattern of column agent with the high density filling channel, there is 60% to 80% certain situation that is occupied by the column agent of channel capacity.According to the filling ratio of chromatographic method and employed column agent type and column agent, the cross sectional shape of the groove shape passage that selection will be set up is so that the sample liquids volume of maintenance per unit passage length is in proper range.Therefore, according to used chromatographic method and condition thereof, with the cross sectional shape of groove shape passage according to channel width (W ₁) and the degree of depth (D ₁) be chosen in the scope of 5 μ m to 5000 μ m, more preferably in 20 μ m to 1000 mu m ranges.Common column passage length and channel width (W ₁) or channel depth (D ₁) between ratio (L/W ₁, L/D ₁) be chosen in 5 to 400 scopes, preferably in 20 to 300 scopes, more preferably in 50 to 300 scopes.

Especially, under situation about using according to automatic sample disposal route of the present invention or automatic sample treatment facility, need sample to be in frozen state to satisfy following condition, the adhesion strength P of sample part (top side) per unit passage length of contact lid part basal surface _TopThe adhesion strength P that surpasses sample part (bottom side and two sides) per unit passage length of contact substrate part side _BottomFor this reason, in order to suppress the relative influence of channel side wall, expect channel depth (D usually ₁) and width (W ₁) between ratio (D ₁/ W ₁) at D ₁/ W ₁In≤1 scope, this ratio is by by the material that calculates substrate portion 103 and the fine disposal route of groove shape passage and definite processing accuracy can suitably be selected.

Under situation about using according to automatic sample disposal route of the present invention or automatic sample treatment facility, when the sample separation that obtains by the lock out operation such as electrophoresis is taken out with the frozen state that continues, the cross sectional shape of groove shape passage can be a rectangle or trapezoidal, the wherein trapezoidal width (W that has than bottom portion of groove _1bottom) width (W of bigger open top _1top) (W _1bottom＜W _1top), it helps taking out sample with frozen state.

For the material of substrate portion 103, for example, suitable use is suitable for the material of fine processing, and for example quartzy, glass or silicon perhaps are selected from and can realize expecting the material of fine processing accuracy such as the high ambroin of polycarbonate, PDMS or PMMA.

In the present invention, in the time will finishing the step of peeling off, substrate portion 103 is elastic deformation not, but lid part elastic deformation, and warp architecture is provided on the stripping borderline; For bending range is minimized, flexibility will be demonstrated but the material that only demonstrates slight elasticity distortion is used for plane cover base 101.

In the present invention, can be through being subject to processing, for example set up therein and be used for the hole that liquid injects, this material aspect insulating property, be good, and have the flexibility of the material that is applicable to plane cover base section 101.For example, a kind ofly can be selected from the acryl resin that comprises PMMA (poly-methyl acrylate) and the fluoropolymer resin that comprises PDMS (dimethyl silicone polymer), even the preferred especially processed but thin thickness easily of using also is not easy the flat material that breaks.For the material that is used for binding resin rete 102 base portions, for example, use PDMS, comprise a kind of in the polyolefin of PTFE (teflon), PP (polypropylene), PE (tygon) and Polyvinylchloride, perhaps polyester.For binding resin rete 102, preferably use than the higher material of elastic properties of materials deformation that is used for plane cover base section 101.

In the present invention, in order to take out by means of such as the separation of electrophoresis and the sample that separates, this sample sticks on the surface of binding resin rete 102 with the frozen state that continues, expectation be the freezing object that can keep adhering to it as the resin of binding resin rete 102 base resins.For the outermost layer of binding resin rete 102, though can adopt the form that the adherent coating that provides some adhesion properties is provided, what expect is that adherent coating adhesion strength when being cooled descends.

In covering microchip itself, the outer shape of substrate portion 103 is a rectangle, and the outer shape that seals the lid part 113 of its end face also is a rectangle.When peeling off and removing lid hermetic unit 113,, part projection from the outer shape of substrate portion 103 can be set at least towards the end that is used to apply external force for the end to lid part 113 applies external force.For example, under the situation of the direction of selecting to peel off and remove lid part 113 along the long limit of the rectangular profile of substrate portion 103, make the outer shape of lid part 113 bigger than the long limit of substrate portion 103 aspect its long limit.When lid part 103 is applied external force, its working point can be set at the part high spot on its long limit.In addition, after finishing the peeling off and remove of lid part 113, when by keeping and, can in described part projection, being provided for supporting the zone of the end of the lid hermetic unit that is separated by the maintenance system from the cover section timesharing that the end face transmission of substrate portion is separated.And, can also select a kind of pattern, wherein the long limit along substrate portion 103 is used as the position that applies external force in the part projection on the short side direction of lid part 113 when peeling off and removing lid part 113.

(being used for electrophoresis liquid is injected into system in the passage that covers microchip)

Constituting the material of the lid part 113 of covering passage top surface in the microchip relatively poor aspect wettability is not rare situation.The kapillary that is made of high wettability material can be fed to all kapillaries from an end of passage by capillarity with electrophoresis liquid, but passage for the madial wall that has poor wettability in the microchip, liquid injection system need be provided, substitute the electrophoresis liquid that utilizes capillarity and inject.Particularly, preferably use this pattern, wherein build-up pressure is poor between the inside of liquid reservoir that is provided at channel end and passage, and utilizes the pressure differential of gained to force to be injected into the passage from the electrophoresis liquid that a liquid reservoir provides.

Because being formed on the passage self that covers in the microchip is closely covered by sealing, drawn when being provided to another liquid reservoir by a liquid reservoir and electrophoresis liquid when the gas in left side, electrophoresis liquid is owing to the pressure differential between them is penetrated in the passage.In this step, when electrophoresis liquid complete filling passage, stop to inject.Utilize in use under the situation of electrophoresis liquid implantttion technique of pressure differential, being used for the operation that electrophoresis liquid injects can carry out automatically by using following structure, wherein: append to one of liquid reservoir by the suction system that is used to draw left side gas; The liquid feeder system with minisize liquid measurement that is fit to injection scheduled volume electrophoresis liquid is connected to another liquid reservoir; And, inject release judgement system constantly two systematic connections got up by determining it automatically.

For automatically determining to inject release judgement system constantly, for example, can use a kind of judgement system, whether it utilize and detect the electrophoresis liquid that the injected this detecting unit of complete filling passage.

When electrophoresis liquid during the complete filling passage,, therefore can observe rapid change from the state of insulation to the predetermined resistance by monitoring each passage resistance value between both ends because electrophoresis liquid itself is the medium with certain electric conductivity.By at this resistance monitoring type detecting unit of electrophoresis liquid that wherein utilized of each passage two ends assembling, can judge the occupied state of electrophoresis liquid as the function of conductive medium.

And electrophoresis liquid is liquid, and its specific inductive capacity is obviously different with gas.By utilizing this feature, can use this monitoring unit, wherein two of the plane capacitance type electrodes are set on two sidewalls of each passage the phenomenon when detecting electrophoresis liquid and be penetrated into space between the electrode, and it causes the change of electric capacity.By assemble the detecting unit of this plane capacitance type at every end of each passage, can judge the occupied state of electrophoresis liquid.

In addition because electrophoresis liquid is liquid, so its with gas significantly different aspect the refractive index.For example, under the situation that substrate portion 103 is made of light transmissive material, when electrophoresis liquid began the covering wall surface, the light reflection that is formed on the conduit wall surface in its end face changed.When providing when utilizing this phenomenon to detect this reflection detecting unit from the light reflection of a wall surface of passage, can determine whether electrophoresis liquid has reached the wall surface part of monitoring of passage.By reflect the liquid detecting unit of monitoring type in every end fit walls surface light of each passage, can judge the occupied state of electrophoresis liquid.

Can by integrated utilization aforementioned liquids detecting unit be used to judge the system of electrophoresis liquid occupied state and the electrophoresis liquid injection system that utilizes pressure differential and determine implant operation finish time is thus automatically realized the automatic operation of whole electrophoresis liquid implant operation.

(being used for fixing system, substrate portion cooling system that the substrate portion of covering microchip is arranged and the control module that is used for cooling system)

When lid part 113 is peeled off and removed to the end face of the substrate portion 103 of microchip, after having fixed substrate portion 103, external force is applied to an end of lid part 113, so that make end forced displacement on the direction that is basically perpendicular to the bonding plane between substrate portion 103 and the lid part 113 of lid part 113.Be accompanied by this displacement of one end, lid part 113 presents flexible structure with respect to the bonding plane.

In the stage that has applied external force, the fixing base part in case substrate portion 103 move.Simultaneously, before the operation of peeling off and removing lid hermetic unit 113, the fluid sample that cools off the electrophoretic separation in the groove shape passage that is present in substrate portion 103 is so that the whole liquid sample is in freezing state.This fluid sample is in following state, and wherein electrophoretic separation is the soluble substance dissolved of electrophoresis liquid, forms spot (spot).Though its solvent composition is a water, buffer composition etc. dissolve wherein, and because the cold point reduction, so its freezing following temperature of freezing point (0 ℃) that starts from.For this reason, expectation be that the whole liquid sample is cooled off fast, down to the temperature of the temperature that significantly is lower than freezing beginning,, make the whole liquid sample in the groove shape passage freezing immediately thus so that make it be in the supercool state once.On the other hand, if aqueous solvent is slowly freezing under the temperature of the temperature that is lower than freezing beginning slightly, freezing other local beginnings with outside spot, this be because, though the concentration of institute's dissolved substance is higher at the spot place, material concentration is low in the zone beyond the spot.In this case, compressed spot not freezing zone on every side by the freezing volumetric expansion that causes, it can cause that liquid leaks out groove shape passage.For fear of this situation, expectation be to be achieved as follows state, wherein the temperature by fluid sample being cooled fast to the temperature that significantly is lower than freezing beginning is so that make it be in the supercool state once, carry out the whole inboard that is chilled in groove shape passage.

Therefore, preferably utilize the substrate portion cooling system, it realizes the supercool state by the quick cooling liquid in bottom surface from the substrate portion 103 of microchip once so that whole passage reaches uniform temperature, and this uniform temperature significantly is lower than the temperature of freezing beginning.The expectation be, cooling system can have following layout, the form that wherein whole bottom of its uniform contact substrate portion, and substrate portion fixed system and substrate portion cooling system integrate is desired.

For fixing of substrate portion, though can use the type of the sidewall sections of fixing base part, the type of this fixing base part bottom surface is preferred.For example, be fit to use this type, wherein, after flat board is processed in the bottom surface of substrate portion, the bottom surface of substrate portion is fixed in the precalculated position on the vacuum chuck system fixed station.Though himself thickness is several mm or littler, because the planar dimension of microchip substrate portion 103 is not little of several mm, its minor face and long limit are almost more than 10cm, but can be not too many greatly, therefore preferably use following pattern, wherein by utilizing such as the cooling device of Peltier device the surface cool of vacuum chuck system fixed station to predetermined temperature.

Incidentally, with the integrated described substrate portion fixed system of substrate portion cooling system in, when the microchip with the sealing of fixed mesa and lid is cooled to significantly to be below the freezing point the temperature of (0 ℃), if surrounding air environment comprises moisture, then it will be condensed and be freezing.In order to prevent to condense, so make up fixed mesa and have the surrounding air environment of covering microchip to make its gas that keeps dry and do not have moisture with freezing.Particularly, will have this structure, be installed in the gas-tight container comprising the zone self of substrate portion fixed system and substrate portion cooling system, and the inside of this gas-tight container remain dry air or drying nitrogen air ambient.

Fluid sample in passage is not under the situation of freezing state, because vibration can constitute the factor that causes liquid mixing in the passage during the transmission, in above-mentioned lock out operation step, the substrate portion 103 of microchip is fixed on the microchip fixed position of this integrated substrate portion fixed system and substrate portion cooling system such as electrophoresis.Integrated substrate portion fixed system and substrate portion cooling system are offered electrophoresis equipment, and, when the lock out operation such as electrophoresis has finished, carry out the rapid cooling of the fixing and substrate portion of microchip substrate portion 103 rapidly by integrated substrate portion fixed system and substrate portion cooling system.

In the lock out operation stage such as electrophoresis, if some other fixed forms are used for the fixing of microchip substrate portion 103, then adopt the pattern that integrated substrate portion fixed system and substrate portion cooling system is transferred to the position of the tight bottom that contacts microchip substrate portion 103.Optionally, when the microchip of lid sealing was set up and was fixed on the precalculated position of equipment before the lock out operation such as electrophoresis, even using this integrated substrate portion fixed system and substrate portion cooling system to fix, also can select following pattern, wherein along with the operation of packing into of the lid sealing microchip that will use can be transmitted integrated substrate portion fixed system and substrate portion cooling system.

For with the whole liquid sample fast cooling down to the temperature of the temperature that significantly is lower than freezing beginning so that make it be in the supercool state once, so that the whole liquid sample in the groove shape passage is freezing in advance, expectation be chilling temperature to be arranged on below the freezing point (0 ℃) at least 10 ℃ to 30 ℃ scope, that is, at least-20 ℃ or lower.When being cooled to described chilling temperature, fluid sample is in the supercool state once, and the whole liquid sample in the groove shape passage takes place freezing in advance thus.In this technology, slight volumetric expansion can take place.Lack at the side surface of passage under the situation of wettability, the turning of the square-section of passage particularly begins to contact the turning of the passage top surface of lid part 113, has not by the zone of the liquid filling of liquid state.Yet, when liquid enters freezing state, reach a kind of state, wherein it begins the basal surface of the lid part 113 of contact channels top surface top.By such as the method for electrophoresis and sample separation, enter and thisly freezingly more preferably be fit to use the present invention with this situation that realizes closely contacting lid part 113 basal surfaces, the basal surface that has wherein utilized lid part 113 with by separate such as the method for electrophoresis, enter the adhesiveness of the sample of frozen state.

Control module by cooling system can carry out automatically and finish sequence of operations under predetermined condition, comprise by the substrate portion fixed system fixedly microchip substrate portion 103, by the substrate portion cooling system come cooling base part 103 and fluid sample in the freezing groove shape passage and the control of temperature subsequently to keep freezing state.

(being used to peel off and remove the system of lid part)

In the present invention, when constituting the substrate portion 103 of covering microchip and lid part 113 when separated, after the substrate portion 103 of having fixed microchip, adopt and peel off and remove the technology of tight adhesion in the lid part 113 of substrate portion 103 end faces.

Particularly, in order to reduce adhesion strength (this adhesion strength has realized making the coherent condition of the predetermined arrangement that the bottom surface of the end face of substrate portion 103 and lid hermetic unit 113 closely is attached to each other), lid hermetic unit 113 is applied external force, this external force has the component on the direction of the substrate portion of being basically perpendicular to 103 end faces, so that crooked lid hermetic unit 113, and peel off with the speed of expectation with the form that upwards rises lid hermetic unit 113 ends, simultaneously keep crooked with predetermined curvature.In the present invention, in the stage of peel-off hd part 113, keep by the coherent condition on the sample top surface that separates such as the method for electrophoresis, the basal surface of this sample contact lid part 113 also is in frozen state in groove shape passage, the result finishes peeling off of lid part 113 with following state, and wherein the sample by the maintenance freezing state that separates such as the method for electrophoresis sticks on the basal surface of lid part 113.

At first, the adhesion strength p of per unit area between the basal surface of top surface by substrate portion 103 and lid part 113 ₀The top surface of substrate portion 103 and the basal surface of lid part 113 are adhered to one another.In this case, because the adhesion strength between the top surface of substrate portion 103 and the basal surface of lid part 113, even on an end of lid part 113 is being basically perpendicular to the direction of substrate portion 103 end faces, rise and lid part 113 when being bent, also exist not begin the scope peeled off.Even under the situation of observing bigger bending, exist and peel off the threshold value of beginning.Curved shape when satisfying threshold condition is limited by δ and L, wherein δ is apart from the displacement of substrate portion 103 end faces at an end place of lid part 113, to be the border that begins to be in contact with one another, the bottom surface from the end face of substrate portion 103 and lid part 113 apply the length of the working point of external force to lid part 113 1 ends to L, and curved shape presents the arc with constant substantially radius of curvature R.Thus, the angle so that θ represents this arc satisfies following equation.

L＝R·θ

δ＝R(1-cosθ)

When bending was in this shape, the power P that is applied to the boundary that the bottom surface of the end face of substrate portion 103 and lid part 113 begins to contact with each other was expressed as follows approx, and wherein d is a thickness, and b is that width and E are effective young's moduluses of lid part 113:

δ＝P·(2L) ³/{4bd ³E}

P＝δ·(4bd ³E)/(2L) ³

When the displacement of lid hermetic unit 113 1 ends 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 radian θ becomes θ → θ+Δ θ ₁, the variation of change is described below:

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 is in this shape, be applied to the power P+ Δ P of the boundary that the bottom surface of the end face of substrate portion 103 and lid part 113 begins to contact with each other ₁Be expressed as follows approx:

P+ΔP ₁＝(δ+Δδ)·{4bd ³E}/(2L) ³

Peel off and obtaining this P+ of reducing Δ P ₁Carry out slightly during → P.Thus, it turns back to the state of peeling off when no longer carrying out.Peeling off when stopping, curved shape presents the arc with constant substantially 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 of the boundary that the bottom surface of the end face of substrate portion 103 and lid part 113 begins to contact with each other ₂Be expressed as follows approx:

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

Thus, when reducing: (P+ Δ P ₁) → (P-Δ P ₂) when taking place, Δ L part is divested.Therefore, reducing of Δ L part adhesion strength: (P+ Δ P corresponding to change ₁) → (P-Δ P ₂).With p ₀The adhesion strength p of per unit area between the end face of expression substrate portion 103 and the bottom surface of lid part 113 ₀:

(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, the distortion (the little bending that radius of curvature R is little) of estimating to surpass the threshold condition that is expressed from the next need remain on the border that the bottom surface of the end face of substrate portion 103 and lid part 113 begins to be in contact with one another:

3·(1/L)·P≈p ₀·b

Thus, the external force that is applied on lid part 113 1 ends is 1/2P, and selects in following scope at least:

(1/2P)＞p ₀·b·L/6

This scope can make to be peeled off between the basal surface of the top surface of substrate portion 103 and lid part 113.

Under situation about using according to automatic sample disposal route of the present invention or automatic sample treatment facility, keep substrate portion 103 and lid part 113 tight adhesion by means of loading enough external force, rather than the adhesion strength of the top surface of basal surface by lid part 113 and substrate portion 103, obtain the maintenance of tight adhesion state between the basal surface of the top surface of substrate portion 103 and lid part 113, wherein this intensity is replenished by external force.At least when reaching aforementioned chilling temperature, the adhesion strength of the top surface of the basal surface of lid part 113 and substrate portion 103 is lower than certain level.Particularly, in this cooling situation, with the basal surface of lid part 113 with by the per unit area adhesion strength p between the top surface of sample that separate, that keep freezing state such as the method for electrophoresis ₁Be provided with greater than the per unit area adhesion strength p between the basal surface of the top surface of substrate portion 103 and lid part 113 ₀(p ₁＞p ₀).In addition, the per unit area adhesion strength p between the top surface of the basal surface of lid part 113 and sample that pass through to separate, the maintenance freezing state such as the method for electrophoresis ₁Greater than groove shape passage with by such as the per unit area adhesion strength p between the method for the electrophoresis sample basal surface that separate, that be in freezing state ₂(p ₁＞p ₂).

In this case, the threshold condition that is used for peeling off between the basal surface of sample top surface that is in freezing state by electrophoretic separation and lid part 113 is expressed as similarly:

3·(1/L)·P≈p ₁·b＞p ₂·b

Radius-of-curvature under this threshold condition is represented as R _Eq2

Thus, in the present invention, select this state, wherein the radius of curvature R of the expression borderline bending of peeling off is not less than the radius of curvature R under the aforesaid threshold values condition _Eq2(R＞R _Eq2), and avoided being in peeling off between the basal surface of the sample top surface that passes through electrophoretic separation of freezing state and lid part 113 thus, simultaneously between the basal surface of the top surface of substrate portion 103 and lid part 113 and groove shape passage and remaining between the basal surface of sample freezing state, by electrophoretic separation peel off.

On the other hand, when the radius of curvature R of the bending of representing lid part 113 basal surfaces changes, particularly when the crooked radius of curvature R of expression reduces suddenly, increase by shear stress (shearing stress) big on the sample that separates such as the method for electrophoresis, and division takes place along the many crackles (granule boundary) in the Frozen Body suddenly.In addition, trouble shown in Figure 1 can take place, just these fragments of splitting are stripped from and are come off.Keep the crooked constant this state of radius of curvature R of expression, therefore, can avoid obscission, and can keep fragment to continue to stick to state on the basal surface of lid part 113 thus.

Integrally construct the external force application system, lid part end-transfer system and lid part end-transfer speed control system, this external force application system provides and applies the function that has perpendicular to the external force of the component of the direction of lid part end upper substrate part end face, lid part end-transfer system Contact Boundary between the bottom surface of the end face that is basically perpendicular to substrate portion and lid part, the end that the transmission cover section is divided on the symmetrical direction that applies with external force on the lid part end, the end-transfer speed that lid part end-transfer speed control system provides control lid part makes it keep the function of radius of curvature R, with the bottom surface of lid part from the step that the end face of substrate portion is peeled off, with predetermined target value, local bending by border, the place of peeling off upper cover part presents this radius of curvature R, for example, can select the structure that describes below.

(first exemplary embodiment)

The stripping system that is used for the lid part shown in Figure 5 is following type, and after the end of vacuum draw lid part, the roller that has predetermined radii by use winds up it.The crooked radius of curvature R of expression lid part becomes and equals the radius of roller, and by keeping speed of wrap constant, the end-transfer speed of lid part also keeps constant.

According to the desired value of the crooked radius-of-curvature of expression lid part, adjust the radius of roller, and speed of wrap is set.

(second exemplary embodiment)

Lid part stripping system shown in Figure 6 is following type, behind the end of clamping the lid part by clamping, with its rise.In this action, select ascending velocity according to the desired value of the crooked radius-of-curvature of expression lid part.

(the 3rd exemplary embodiment)

Lid part stripping system shown in Figure 7 is the type that rises lid part one or both ends.The boost bottom surface of lid part of the clamping unit that is used for the one or both ends of mobile lid hermetic unit.In this action, select ascending velocity according to the desired value of the crooked radius-of-curvature of expression lid part.

(the 4th exemplary embodiment)

Lid part stripping system shown in Figure 8 is following type, after the end of clamping the lid part by vacuum draw unit, rises it.In this operation, select ascending velocity according to the desired value of the crooked radius-of-curvature of expression lid part.

The vertical moving speed of the rotation angle by utilizing the rising arm and the pillar of supporting rotating shaft is adjusted the control of ascending velocity in the scope of expectation.

(the 5th exemplary embodiment)

Lid part stripping system shown in Figure 9 is following type, after the end of clamping the lid part by vacuum draw unit, rises it.In this operation, select ascending velocity according to the desired value of the crooked radius-of-curvature of expression lid part.

In some cases, the platform of fixing base part can reduce so that substrate portion is risen relatively.

(the 6th exemplary embodiment)

Lid part stripping system shown in Figure 10 still is following type, after the end of clamping the lid part by vacuum draw unit, rises it.In this action, select ascending velocity according to the desired value of the crooked radius-of-curvature of expression lid part.

In some cases, the platform of fixing base part can reduce so that make cover section divide rising relatively.

(the 7th exemplary embodiment)

Lid stripping system shown in Figure 11 is following type, after insert the end of lid part, moves this rail unit at the spade rail unit with pre-determined tilt angle in the end that tilts to rise the lid part along this.In this action,, divide crooked radius of curvature R by selecting translational speed to control the expression cover section according to the desired value of radius of curvature R.

Particularly, the radius of a circle that connects this inclined-plane and substrate portion end face in constitutes the crooked radius of curvature R of expression lid part.The crooked radius of curvature R of expression lid part reduces along with the increase of translational speed.When translational speed fixedly the time, adjust the radius of curvature R that reaches the expression bending of determining by this condition.

(disassembling system that is used for institute's separate cover subdivision)

In the present invention, finish peeling off of lid part 113 with following state, wherein will remain on frozen state pass through remain on the lip-deep state of the back of the body that adheres to lid part 113 such as the sample that the method for electrophoresis is separated.After this, the lid part that maintenance is separated for example in above-mentioned second embodiment, holds it in the state of clamping lid part end by clamping unit, and by the transmission clamping unit its top surface from substrate portion is removed.And, in the 4th to the 7th embodiment, by its top surface from substrate portion being removed with the state transfer that remains in the system that is used to divest.In the 3rd embodiment, can adopt this pattern, wherein with the state that the end is remained on clamping unit the lid part of being separated is removed from the end face of substrate portion by independent clamping unit.

In the present invention, step in peel-off hd part 113, keep coherent condition in groove shape passage, entering on top surface freezing state, by the sample that separates such as the method for electrophoresis, do not carry out the divesting of wall surface of groove shape passage, the result finishes peeling off with the state that passes through to stick to lid part 113 basal surfaces such as the sample that the method for electrophoresis is separated that will remain on frozen state of lid part 113.Therefore, remove with the lid partial fixing that separated and from the top surface of substrate portion, top surface and the basal surface with the lid part spins upside down so that upwards expose the basal surface of lid part then.For turn over function, for example, in a second embodiment, clamping unit is removed when state is clamped in the clamped unit, end that keeps the lid part, thereby can be finished dismounting and upset from the substrate portion top surface.And, in the 4th embodiment, when keeping holding the state of lid part by the system that is used to peel off, finish the rotation of the system that is used to peel off, thereby can finish upset, and can carry out from the dismounting of substrate portion top surface simultaneously.In the 5th and the 6th embodiment, can adopt this pattern, wherein hold system and be subjected to turning operation, keep clamping the state of lid part end simultaneously with vacuum draw unit.In the 3rd embodiment, the lid part of being separated is removed from the end face of substrate portion by independent transmission clamping unit with the state that clamped unit, its end is clamped, thereby can be finished from dismounting of substrate portion top surface and upset.

(being used for segmented system) by the sample that separates such as the method for electrophoresis

In the present invention, the top and bottom surface of lid part is spun upside down, so that be achieved as follows layout, wherein sticking to the sample that passes through to separate such as the method for electrophoresis lid part basal surface, that be in lasting frozen state exposes from the teeth outwards, after this, can be segmented into a plurality of fragments along passage with being in the sample that passes through to separate that continues frozen state such as the method for electrophoresis.

Particularly, when the basal surface of lid part is crooked with convex shape, follow this bending and the shear stress that obtains be loaded in stick to lid part basal surface, be on the Frozen Body that passes through the sample that separates such as the method for electrophoresis that continues frozen state.In this state, when the surface that makes sharp-pointed blade clevis tool contact refrigeration attitude so that it is applied slight stress, thereby at this some place cracking appears just.Frozen Body repeats the cracking operation with predetermined space, so that can be segmented into a plurality of fragments along passage.When the fragment of Frozen Body is separated by cracking in advance, by the basal surface of fragment from the lid part being split with basal surface than the crooked lid part of the convex of small curvature radius.Therefore, can the individual chip of Frozen Body be split from lid basal surface partly by using aforesaid way, so that each fragment is collected in each hole of the segmented plate (porous sample panel) that comprises a plurality of holes.

In the present invention, can also construct the automatic sample treatment facility that is associated with this segmented system.

In addition, collect each fragment of solidifying attitude in the fragment plate hole (porous sample panel) by the batch operation of front and stand again dissolution process comprises protein of being separated in the individual chip etc. with preparation fragment sample liquids.Also preferably select following pattern, system and the segmented system that wherein will finish distribution and dissolution process more subsequently are assembled in the automatic sample treatment facility.

The operation that is included in the single step in the sequence of operations of front can oneself be carried out automatically, and can should the series operation form technology fully automatically by using automatic operation control system, this automatic operation control system has the function that automatically performs the work of each system according to predetermined process.

(analytical approach that is used for biological sample)

In the analytical approach that is used for biological sample according to the present invention, using equipotential to focus under the situation as the electrophoresis operation, can will be comprised in multiple proteins in the fluid sample of wanting analyzed to keep its natural preservation state and to keep its active state to separate.By utilizing its advantage, can serve as that the basis checks whether comprise the protein that presents targeted activity with the biological assay analysis result to each fragment by the sample that separates such as the method for electrophoresis.For example, combine by chemically examining with biological sample analysis method according to the present invention, can be used as " preliminary screening (primary screening) " mode, be used for checking whether the protein component that demonstrates given activity is comprised in molecular wt and the isopotential point of wanting analyzed fluid sample and further determining to demonstrate the target protein composition of given activity.

(being used for the chemico-analytic equipment of microchip)

The preferred one-piece construction that is used for the microchip chemical analysis apparatus according to of the present invention will be further described.

Especially, microchip chemical analysis apparatus according to the present invention is applied to handle the sample that causes the electric conductivity separation between atom, wherein, as target sample, the lock out operation such as the expectation of electrophoresis of the passage of wanting analyzed fluid sample experience to be formed on to cover in the microchip by use, each that makes a plurality of materials of being included in the fluid sample is thus separated on the position so that form point along passage, but can also use this equipment when using some other chemical analysis technologies that cause the electric conductivity separation between excepting that atom.

In this case, a kind of hardware configuration of integral body is proposed, comprise the chemical analysis unit 1 that is used at the passage sample for chemical analysis of microchip, the solution fixed cell 2 that is used for fixing sample for chemical analysis and electrophoresis liquid and lid part separative element 3 in this structure, this lid part separative element is used for lid part is separated so that the sample of fix the passage with the substrate portion of microchip partly collects with lid from substrate portion.To with system or other single parts of structures of samples assembling in can the chemical analysis microchip or be used for below using with subsequent stage that described sample carries out analyzing and the spare system that adopts without limits, as long as they do not influence the analysis of chemical analysis unit 1.

Though be not particularly limited, the chemical analysis of finishing by chemical analysis unit 1 among the present invention for example comprises phoretic separation, and allows the equipotential of single isopotential point place sample concentration to focus on particularly suitable.In this case, chemical analysis unit 1 can be made of electrode part and phoretic power supply.Provide voltage to the electrode part by distribution from phoretic power supply, and by use the electrode part with voltage be applied in the microchip passage electrophoresis liquid so that electrophoresis take place.Can also arrange on the lid part of microchip that further the liquid reservoir cover unit is to suppress electrophoresis evaporation of liquid in the passage.And, can also be provided at and apply the electric current monitor that is used for the monitor current level during the voltage.

Chemical analysis unit 1 can also provide transmission system, be used for the liquid reservoir cover unit and/electrode part is transferred to their precalculated position automatically.Can be used alone or in combination one or more this spare systems.

For the solution fixed cell 2 among the present invention, though be not particularly limited, for example use cooling system, this cooling system is fixed by freezing sample or by described chemical analysis unit 1 chemico-analytic electrophoresis liquid.

What expect is that the cooling system among the present invention is a type of cooling off the microchip substrate portion by direct contact.Can have only a cooling system or a plurality of this systems are arranged, and it can add in the system of the substrate portion side with second cooling system, this second cooling system provides cooling by the liquid reservoir cover unit from lid part side.Available cooling system for example includes, but not limited to, and uses the cooling system or the refrigeratory of Peltier device.

Thereby thereby lid part separation vessel 3 used in this invention has the transmission system that attracts system, the pneumatically of contact or fixed cover part to attract the system of contact or fixing base part and fixing lid part and substrate portion are separated from each other by vacuum chuck.

Though be not particularly limited, thereby but passing through of using among the present invention system that vacuum chuck attracts contact or fixed cover part can be make the lid part by vacuum chuck attracted to fixed system pump unit, lid partly is adhered to the adhesion unit 12 of fixed system, perhaps make cover section divide contact or be fixed to the lid part fixed cell of fixed system.

Though be not particularly limited, thereby the system that passes through vacuum chuck attraction contact or fixing base part that uses among the present invention for example can be the substrate portion fixed cell that makes substrate portion be attracted to the substrate portion absorbing unit of fixed system, substrate portion is adhered to the substrate portion adhesion unit of fixed system or makes the substrate portion contact or be fixed to fixed system by vacuum chuck.

The lid part vacuum draw unit or the substrate portion vacuum draw unit that can be used among the present invention have aspirating hole and reduce the depressurized system of pressure by aspirating hole, and can be by the object of vacuum chuck attraction near aspirating hole.

Though be not particularly limited, make the transmission system of using among that fixing lid part and substrate portion are separated from each other, the present invention for example can be the chip platform unit that moves up and down substrate portion or lid part, the lid part is twined in upset roller, clamp or hook the clamping unit of lid part or substrate portion or hook the unit or in ON/OFF unit that the axostylus axostyle as rotation center opens or closes on every side.

Expectation be that microchip chemical analysis apparatus of the present invention is further provided with by connecting the lid part-substrate portion connected system construct microchip and the solution injected system that is used for sample and/or electrophoresis liquid are injected into the microchip passage.It also provides detecting signal unit, is used to detect the chemical analysis process or the result that carry out in microchip.

Though be not particularly limited, but the lid part-substrate portion connected system that uses among the present invention for example can be with the positioning guide rail such as projection, depression, hole or wedge angle of the form fit of microchip design, be used to keep the retainer or the transmission system of microchip, and this transmission system is by being arranged in precalculated position with the cover section branch substrate portion and pressing substrate portion to be connected substrate portion and lid part with the lid part to increase the adhesion tightness.Can be used alone or in combination one or more this systems.

Though be not particularly limited, the solution injected system of using among the present invention for example can be depressurized system or compression system, and it introduces solution in generation pressure differential between the opening at place, microchip passage two ends.

Though be not particularly limited, the detecting signal unit that uses among the present invention for example can provide light irradiation unit.Detecting signal unit has photodetector at least, is used for the measuring light wavelength signals, for example absorbing wavelength or fluorescence.For example, use exciting light irradiation tunnel from light irradiation unit, and by using photodetector to detect fluorescence.Can when using chemical analysis unit 1 analytic sample or after analysis by using solution fixed cell 2 that solution is used this detecting signal unit after fixing.

Microchip chemical analysis apparatus of the present invention can be constructed by the combination of adopting aforementioned a kind of pattern or various modes.

Consider easy operation, microchip chemical analysis apparatus preferably of the present invention is further provided with control module.Voltage by using the current monitoring unit to come the monitor current level to be provided by power supply with control can be provided control module.And control module can be used for determining chemico-analytic terminal point according to the current level monitored, duration that voltage applies, and be used to control the operation of cooling system.And, thereby thereby thereby control module can also be used to control by vacuum chuck attract contact or fixed cover part system, attract the system of contact or fixing base part and with the be separated from each other operation of the transmission system of exposing passage of fixing lid part and substrate portion by vacuum chuck.And, control module can be used for the used transmission system of lid part-substrate portion connected system of control linkage substrate portion and lid part, use depressurized system or the compression system that produces different pressures in the control solution injected system, control the heating system or the depressurized system that use in the drying system, and be used for checking the sample analysis state of signal checking unit.

Microchip chemical analysis apparatus according to the present invention can have the structure that is further provided with the unit that is used for the lid partial switching.Thereby lid part overturn system is the unit that spins upside down the lid part by the lid part of using 3 rotations of lid separation vessel to separate from substrate portion.

In this case, in its whole hardware configuration,, fix electrophoresis liquid or sample by using solution fixed cell 2 by after using the sample in the chemical analysis unit 1 chemical analysis passage.Solution fixed cell 2 freezing and fixed sample and electrophoresis liquid as cooling system.Then, by using lid part separative element 3 that the lid part is separated with substrate portion.After the freezing sample that will expose and electrophoresis liquid adhere to lid part one side, by using the lid partially switched cell upwards can be overturn in the surface that adheres to freezing sample and electrophoresis liquid and keeping this direction.Preferably, when the timesharing of lid partially switched cell contact cover section,, before its contact cover section is divided, the lid partially switched cell is cooled off in order to prevent to adhere to the freezing sample and the dissolving of electrophoresis liquid of lid part.

In the present embodiment, for freezing sample and electrophoresis liquid are adhered to a lid side partly, need to select the material of microchip substrate portion, structure and surface property, the material of lid part and the composition of surface structure and solvent of passage.

For freezing sample and electrophoresis liquid being adhered to a side of lid part, for example, can reduce the wall surface of passage and the frictional work between the frozen soln, solidify solution and come off from passage easily so that make.The mode that reduces to rub comprises uses semicircle or the inverted triangle cross sectional shape as passage.And, can so construct substrate portion, make the total area of the projection on the whole circumference of wall surface of the passage on the plane of the upside of the wall surface that is orthogonal to passage be not more than 0.5 times of surface area that cover section is divided solution in the contact passage.The total area of projection is meant on perpendicular to the whole circumference of the wall surface of the passage on the plane of the wall surface upside of passage, for example the limit of the wall surface of passage tilt or situation perpendicular to top surface under, when wall surface protrudes into plane perpendicular to the passage top surface along the periphery of bulge-structure, the area that obtains by the integration of the protruding area of the wall surface of passage.By making its maintenance be not more than 0.5 times of surface area that cover section is divided solution in the contact passage, can reduce the contact area between the wall surface of solution in the passage and passage, and therefore can reduce frictional resistance.Optionally, the frictional resistance between the wall surface of solution in the passage and passage also can reduce by the surface energy that reduces channel surface.In order to reduce the surface energy of channel surface, can use the material of low, smooth plastic resin, silicones, fluorine resin, acryl resin, polypropylene or the polyolefin that is shaped of surface energy as substrate portion.In addition, under the situation of using the high substrate portion material of surface energy, can use the surface of the lower coated materials passage of aforementioned surfaces energy.

For it being adhered to a side of lid part, for example, lid stock can be selected from high metal of surface energy or glass, and perhaps the surface of lid part can have fine roughness with the surface of increase lid part and the frictional resistance between the solution.

Then, will explain according to microchip chemical analysis apparatus of the present invention with reference to more specific example.Incidentally, technical scope of the present invention is not limited to these specific embodiments.

(the 8th embodiment)

Fig. 3 schematically shows the sketch map that is used to carry out the equipment that equipotential separates as microchip chemical analysis apparatus exemplary embodiment of the present invention.In this 8th embodiment, separate to come sample for chemical analysis by equipotential; By cryofixation with sample and electrophoresis liquid fixing after, the separate cover subdivision also adheres to the sample of cryofixation and electrophoresis liquid on the side that lid partly exposes.

Microchip is made of substrate portion 103 with channel architecture and the lid part 113 that has as the pore structure of liquid reservoir.

At first, substrate portion 103 is installed on the chip platform along the chip guide rail.The chip platform comprises Peltier device, aspirating hole and transmission system.The Peltier device is also with the cooling system that acts on the cooling microchip.Aspirating hole is connected to vacuum pump, and substrate portion 103 is fixed to the chip platform by vacuum chuck.Transmission system is with acting on the transmission system that lid part and substrate portion are separated from each other.It also is used in the lid part-substrate portion connected system.

Then, lid part 113 is installed on the lid platform along the lid guide rail.The liquid reservoir cover unit is provided with the aspirating hole in electrode part and the bottom surface, and electrode partly is disposed in the reservoir part of lid part 113.Aspirating hole is used for reducing pressure by aspirating hole and comes vacuum to clamp liquid reservoir cover unit and lid part 113.The liquid reservoir cover unit provides Peltier device, and this Peltier device is used as the cooling system of lid part when the liquid reservoir cover unit is separated together with lid part 113.And, the liquid reservoir lid partly provides transmission system, it is as being transferred to the transmission system in precalculated position with the liquid reservoir cover unit, has and the transmission system identical functions that lid part and substrate portion are separated from each other, and is used as lid part-substrate portion connected system.

Then, use transmission system upwards to rise the chip platform that substrate portion 103 has been installed on it, so that substrate portion 103 is pressed on the lid part 113, thus by connecting the structure microchip.After this, the position that keeps the chip platform.The liquid reservoir cover unit is removed to expose the liquid reservoir of microchip on lid part 113.The electrophoresis liquid that has wherein dissolved sample is injected in the liquid reservoir of lid part.Particularly, focus in order to carry out equipotential, the ampholyte with 2% (pharmalyte) is as electrophoresis liquid.When the whole passage of microchip is filled with electrophoresis liquid, the electrophoresis liquid that is retained in the liquid reservoir is removed.Then, catholyte and anolyte are injected in the liquid reservoir at place, passage two ends, and the liquid reservoir cover unit is installed on the lid part 113 once more.

So far all transmission systems of mentioning can be operated by using control module.

Measure voltage and the anode of electrode part and the current level between the negative electrode that is applied to the electrode part from power supply by distribution by using the current monitoring unit.Because current level descends in the voltage application time gradually, if therefore can measure the terminal point that its current level or wattage just can determine that equipotential is separated.

At the equipotential after separating, operation is used for the cooling system of chip platform and liquid reservoir cover unit so that freezing sample and/or electrophoresis liquid.Then, when the vacuum chuck by aspirating hole attracts chip, substrate portion 103 is pressed on the lid part 113, the chip platform is descended so that lid hermetic unit 113 is separated with substrate portion 103 by the chip platform that raises once.By from top and below lid part 113 is pushed with guide rail, the liquid reservoir cover unit is come work as lid part stationary installation.Have the substrate portion 103 of chip platform cooling system by continuous cooling, can on substrate portion 103, expose freezing sample and/or electrophoresis liquid.Point at this moment is positioned at substrate portion 103 under the lid part 113.By using the lid roll-over unit to spin upside down lid, the liquid reservoir cover unit keeps wherein attracting by the vacuum clamping of aspirating hole the state of lid part 113 simultaneously.

In this case, glass is used for substrate portion 103, and selects the channel depth of channel width and the 10 μ m of 100 μ m.Silicones is used for lid part 113.In this case, by substrate portion 103 is pressed on the lid part 113, freezing sample and electrophoresis liquid can stick on lid one side more firmly.

And, silicones is being used under the situation of substrate portion, it is chosen as the channel depth of channel width and the 40 μ m of 400 μ m, and glass is used for lid part 113, freezing sample and electrophoresis liquid can stick on lid one side 113 securely, and substrate portion 103 are not pressed on the lid part 113.

Because freezing sample and electrophoresis liquid have solid-state and easy to handle advantage more thus, therefore can freezing sample and electrophoresis liquid be divided into fragment separately and they are transferred to are used for the further precalculated position of processing.

Industrial applicibility

Can be with the repeatability that has sample treatment, automatic sample disposal route of covering microchip, using it and the use that is used to analyze to have the automatic sample treatment facility that covers microchip to be used to strengthen to be used for the sample preparation steps of further analyzing that is used to analyze according to the present invention, should further analyze and use the sample of having handled by electrophoretic separation, this is further analyzed for example is biological assay and chemical assay.

Claims

1. a microchip comprises substrate part and lid part, disposes the groove shape passage that is formed in the substrate part, it is characterized in that:

The cross sectional shape that is formed on the groove shape passage in the substrate portion of microchip of lid is trapezoidal or top and the following rectangle that equate of top than the following length of side; And

When cooling microchip during, at the sample of frozen state and the lid cohesive strength p of the per unit passage length of the part (top) that contacts of the basal surface of (top) partly with the fluid sample in the freezing passage _TopThe cohesive strength p that has surpassed the per unit passage length of the part that sample contacts with the side wall surface of substrate portion (following and two sides) _Bottom

2. method, by utilizing the passage that forms in the microchip of lid that has as claimed in claim 1, thereby using predetermined isolation technics makes after the lock out operation that analyzed fluid sample has been stood to expect, this method is used for handling the fluid sample of the separation that remains on passage, described passage is formed in the microchip of lid, it is characterized in that:

The described microchip that covers has following structure, the groove shape passage that wherein is formed in its substrate portion has reached the state that bonds together with predetermined structure with the lid part that seals this substrate portion top surface, so that make the top surface of substrate portion and lid basal surface partly bonded to one another securely

Be formed at the passage that covers in the microchip by utilization to after analyzed fluid sample is finished the lock out operation of expectation, said method comprising the steps of:

Cooling step, wherein make the fluid sample that has separated that remains in the passage be subjected to following refrigeration operation, this refrigeration operation describedly has the substrate portion of covering microchip so that realize freezing point or predetermined low temperature level condition below freezing by cooling off, thereby makes the hydrosolvent that comprises wherein freezing;

The step of peel-off hd part, its purpose is, by having the substrate portion of covering microchip to keep chilled under the predetermined low temperature with described, and makes sample leave groove shape passage with the state that is bonded to lid part basal surface, simultaneously the sample that is separated is remained on lasting freezing state

The operation of lid partly being peeled off and removing from substrate portion is carried out, so that keeping and predetermined threshold R so that the basal surface of lid part is peeled off with the top surface of substrate portion in end by external force being applied to lid part _Eq2Discharge the operation of cohesive strength in the time of relevant condition, this cohesive strength closely contacts the basal surface of the top surface of substrate portion and lid part each other and reaches bond state with predetermined structure, wherein with predetermined threshold R _Eq2Relevant condition is meant that the radius of curvature R that is demonstrated by the local bending of the interface upper cover part at the place of peeling off is greater than described threshold value R _Eq2(R＞R _Eq2); And

The step of separate cover subdivision, wherein, after described strip step finishes, transmit as follows and turning operation, promptly, to partly be transmitted top surface by discharging with the bonding of the top surface of substrate portion and the fixing lid that separates away from substrate portion, keep a kind of state simultaneously, the state that described sample separation is kept freezing state and kept boning with lid basal surface partly under this state, top surface and basal surface with the lid part turns reversedly then, and the cover section branch that separates remained following structure, the sample separation of the basal surface that is bonded to the lid part of keeping freezing state is exposed in its surface;

Wherein these series of steps are carried out in turn.

3. method, by utilize as claimed in claim 1 have cover the passage that forms in the microchip and use after thereby predetermined isolation technics makes the lock out operation that analyzed fluid sample has been stood expectation, this method is used for handling automatically the separating liquid sample that remains on passage, described passage has been formed on and has covered in the microchip, it is characterized in that:

The described microchip that covers has following structure, in this structure, the groove shape passage that is formed in its substrate portion has reached the state that bonds together with predetermined structure with the lid part that seals this substrate portion top surface, so that make the top surface of substrate portion and lid basal surface partly bonded to one another securely

The step of peel-off hd part, its purpose is, by having the substrate portion of covering microchip to keep chilled under the described predetermined low temperature with described, and makes this sample leave groove shape passage with the state that is bonded to lid part basal surface, simultaneously the sample that is separated is remained on and continue freezing state

The operation of lid partly being peeled off and removing from substrate portion is carried out, so that keeping and predetermined threshold R so that the basal surface of lid part is peeled off with the top surface of substrate portion in end by external force being applied to lid part _Eq2Discharge the operation of cohesive strength in the time of relevant condition, this cohesive strength closely contact the basal surface of the top surface of substrate portion and lid part each other and reaches bond state with predetermined structure, this and predetermined threshold R _Eq2Relevant condition is meant that the radius of curvature R that is demonstrated by the local bending of the interface upper cover part at the place of peeling off is greater than described threshold value R _Eq2(R＞R _Eq2); And

The step of separate cover subdivision, wherein, after described strip step finishes, transmit as follows and turning operation, promptly, to partly be transmitted top surface by discharging with the bonding of the top surface of substrate portion and the fixing lid that separates away from substrate portion, keep a kind of state simultaneously, under this state, the state that described sample separation is kept freezing state and kept boning with lid basal surface partly, top surface and basal surface with the lid part turns reversedly then, and the cover section branch that is separated remained following structure, the sample separation of the basal surface that is bonded to the lid part of keeping freezing state is exposed in its surface;

Wherein these series of steps are carried out automatically.

4. device, by utilize as claimed in claim 1 have cover the passage that forms in the microchip and use after thereby predetermined isolation technics makes the lock out operation that analyzed fluid sample has been stood expectation, this device is used for handling automatically the separating liquid sample that remains on passage, described passage has been formed on and has covered in the microchip, it is characterized in that:

This device is included as the following system of covering microchip configuration, described have to cover be formed on the passage that covers in the microchip by utilization in the microchip and finished the lock out operation that analyzed fluid sample is desired:

The system that is used for cooling base part, this system are suitable for the substrate portion contacting structure of covering microchip to be arranged and install with described;

The control module that is used for cooling system, thereby this unit can remain on freezing point or predetermined low temperature level condition below freezing by cooling to major general's substrate portion of substrate portion cooling system, described substrate portion cooling system is installed to be and the contacted structure of substrate portion;

The system that is used for fixing substrate portion, this system can fix the described substrate portion of covering microchip that has with the structure that contacts described substrate portion cooling system;

Be used to apply the system of external force, this system has the function that the end of lid part is applied external force, this external force has the component on the direction that is basically perpendicular to the substrate portion top surface, so that release cohesive strength, described cohesive strength make the top surface of substrate portion closely contact the bond state that also reaches predetermined structure thus each other with the basal surface of lid part;

Be used to transmit the system of lid part end, this system can be when being applied to external force by described external force application system the end of lid part, the end of transmission cover section branch on the perpendicular substantially direction of the contact interface between the basal surface of the top surface of basic and substrate portion and lid part;

The system that is used for the speed of control transmission lid part end, this system has the function of the transmission speed of control lid part end, so that by use external force application system and lid part end-transfer system with the basal surface of lid part from the technology that the top surface of substrate portion is peeled off, this system works to the end of described lid part simultaneously, the radius of curvature R that is demonstrated by the local bending of the part of the lid on the interface at the place of peeling off keeps under the following conditions, that is, with predetermined threshold value R _Eq2Relatively, radius of curvature R is greater than described threshold value R _Eq2(R＞R _Eq2);

Be used to dismantle the system of separated lid part, this system has following function, with lid part after the EO that the top surface of substrate portion is peeled off, this system keeps by discharging the fixing and lid part that separate from the substrate portion top surface of bonding, with the top surface of its transmission away from substrate portion, and subsequently lid top surface and basal surface partly overturn reversedly, so that the basal surface of lid part is upwards exposed; And

This device further comprises and is used to control its automatic operated system, and this system has the function that the action that makes each system that finishes set sequence of operations is finished automatically according to predetermined process.

5. method that is used for analysis of biological samples, this method is, by utilize as claimed in claim 1 have cover the passage that forms in the microchip and use after thereby predetermined electrophoretic techniques makes the electrophoretic separation operation that analyzed fluid sample has been stood expectation, outside the fluid sample of the electrophoretic separation that in being formed on the passage that covers in the microchip, keeps, the component substances that point separates on described passage is segmented into a plurality of fragments along passage, and subsequently the component substances that is included in the some separation in the described fragment is carried out the biological assay analysis, it is characterized in that:

This method comprises:

Collect step, wherein, utilization be formed on the passage that cover in the microchip finished the electrophoretic separation of the expectation of analyzed fluid sample operated after,

According to the method that the automatic sampling processing of covering microchip is arranged that is used to analyze as claimed in claim 3, peel off and remove by sealing the lid part of covered substrate part top surface securely, and subsequently,

By will be in the groove shape passage of sample from be formed at substrate portion isolating and hold it in simultaneously under the state that is bonded to lid part basal surface, thus collect remain on continue under the freezing state by the sample of electrophoretic separation;

Division step wherein, is segmented into a plurality of fragments with the sample that remains on the electrophoretic separation that continues frozen state along described passage, and

Make the component substances that is separated into spot in the groove shape passage in being formed at substrate portion be comprised in in described a plurality of fragment any one;

Fragment is the step of dissolution process again, wherein each the sample segmentation under the freezing state stands dissolution process more respectively, thereby the sample liquids for preparing each segmentation, wherein each sample fragmented packets is contained in in described a plurality of fragment any one, and described a plurality of segments are corresponding to the sample of the described electrophoretic separation of part;

Identify the step of each sheet segment limit, for along being formed at each of a plurality of fragments that the groove shape passage in the substrate portion cuts apart, based on passage on the positional information at fragment two ends, carry out identification with the corresponding electrophoresis exponential quantity in fragment two ends;

Be used for biological assay and analyze the step of fragment, wherein make the sample liquids of each fragmentation be subjected to the biological assay analysis, whether be contained in the fragment with any component substances of determining to demonstrate particular characteristics that can be by biological assay analysis identification; And

Be used to analyze the step of data, wherein based on the biological assay analysis result of fragment, determine to demonstrate the component substances of analyzing discernible particular characteristics by described biological assay whether in the sheet segment limit of just studying, be divided into mottled, and

Information about the electrophoresis exponential quantity scope of discerning each sheet segment limit, wherein determine to have obtained component substances along groove shape passage based on this information, this component substances demonstrates can be by the special characteristics of described biological assay analysis identification, and it is separated mottled that this component substances is confirmed as wanting.

6. method as claimed in claim 3 is that equipotential focuses on by the lock out operation of using predetermined isolation technics wherein.

7. method as claimed in claim 4 is that equipotential focuses on by the lock out operation of using predetermined isolation technics wherein.

8. method as claimed in claim 5 is that equipotential focuses on by the lock out operation of using predetermined isolation technics wherein.

9. automatic sampling processing method is characterized in that:

According to as claimed in claim 3 be used to analyze the automatic sampling processing method of covering microchip arranged, after the step of dismounting lid part is finished,

Described method further comprises,

Division step, wherein, by isolating in the groove shape passage of sample from be formed at substrate portion and hold it in simultaneously in the state that is bonded to lid part basal surface, thereby collect that predetermined isolation technics is separated by using, be maintained at the sample that continues in the freezing state, sample after will separating along described passage subsequently is segmented into a plurality of fragments

Make the component substances that is separated into spot in the groove shape passage in being formed at substrate portion be comprised in in described a plurality of fragment any one thus;

Fragment is the step of dissolution process again, wherein each the sample segmentation under the freezing state stands dissolution process more respectively, thereby the sample liquids for preparing each segmentation, wherein said sample be comprised in the corresponding a plurality of segmentations of the part of described sample separation in any one in.

10. automatic sampling processing device is characterized in that:

Except according to the system that the automatic sampling processing device that covers microchip is arranged that is used to analyze as claimed in claim 4,

Described device further comprises,

The system that is used for segmentation, it has the function that the sample under the lasting freezing state is cut to a plurality of segmentations, wherein, by isolating in the groove shape passage of sample from be formed at substrate portion and hold it in simultaneously under the state that is bonded to lid part basal surface, thereby collect by using the sample that predetermined isolation technics is separated, this sample is maintained at and continues under the freezing state, and

Be used for dissolving again the system that fragment is handled, it has distribution function and has heat and dissolves function again, each of a plurality of sample segmentations under the wherein lasting freezing state is assigned to each hole of porous sampling plate, and each sample segmentation stands dissolution process more subsequently, thereby the sample liquids for preparing each segmentation, the described sample segmentation that wherein continues under the freezing state is to utilize the described system that is used for segmentation to prepare by the sample that separates is carried out segmentation.

11., be that equipotential focuses on wherein by the lock out operation of using predetermined isolation technics according to the method for claim 9.

12., be that equipotential focuses on wherein by the lock out operation of using predetermined isolation technics according to the method for claim 10.