CN105008052B - The manipulation of droplet size in digital micro-fluid system - Google Patents

The manipulation of droplet size in digital micro-fluid system Download PDF

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Publication number
CN105008052B
CN105008052B CN201380074039.1A CN201380074039A CN105008052B CN 105008052 B CN105008052 B CN 105008052B CN 201380074039 A CN201380074039 A CN 201380074039A CN 105008052 B CN105008052 B CN 105008052B
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electrode
container
array
drop
electrod
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CN105008052A (en
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M·N·费格林
J·马莫
M·B·弗兰克林
T·F·李
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Tecan Trading AG
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Tecan Trading AG
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B01L3/502792—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics for moving individual droplets on a plate, e.g. by locally altering surface tension
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5029—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures using swabs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0427—Electrowetting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/0241—Drop counters; Drop formers
    • B01L3/0268—Drop counters; Drop formers using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/52—Containers specially adapted for storing or dispensing a reagent
    • B01L3/527—Containers specially adapted for storing or dispensing a reagent for a plurality of reagents

Abstract

A kind of droplet manipulation instrument (20) includes：At least one electrod-array (21) is used to cause drop (19) mobile by electricity moistening；Substrate (22) supports at least one electrod-array (21)；And control unit (23) comprising at least one electrode selector (34) being connect at least one voltage control (29).At least one electrode selector (34) is implemented as being individually chosen each electrode (35) of at least one electrod-array (21) and provides the voltage controlled by voltage control (29) to selected electrode (35).Control unit (23) further includes central processing unit (36), for the central processing unit (36) for coordination electrode selector (34) and voltage control (29) to be individually chosen at least one electrode (35) and provide individual voltage pulse at least one selected electrode (35), it includes driving voltage, ground voltage and the group for stopping voltage which, which is selected from,.Control unit (23) can be by the path of the guided movement of the large volume of liquid portion (19') for the more than one electrode (35') for limiting one electrod-array (21) of covering by substantially simultaneously selecting two or more subsequent driving electrodes (35') groups of the electrod-array (21), and selectes in driving electrodes (35') each to these and provide drive voltage pulses along the path.Control unit (23) is implemented as substantially simultaneously providing ground connection to adjacent or identical two or more electrodes (35) the group with pulsed drive electrode (35') or stops voltage pulse.

Description

The manipulation of droplet size in digital micro-fluid system
Related application
The application is on March 4th, 2013 to submit and be disclosed as 2013/0175169 Al's of US on July 11st, 2013 US 13/784,168 corresponds to application, and US 13/784,168 is incorporated into herein in entirety by reference.US 13/784,168 It is the continuous application part for the patent application US 13/139,647 for being disclosed as 2011/0290647 Al of US, patent application US 13/139,647 is the American National stage of the International Application Serial No. PCT/EP09/67240 submitted on December 16th, 2009, international Shen Please PCT/EP09/67240 the excellent of U.S. Provisional Application 61/138,294 and Swiss Patent application number CH 01979/08 is claimed It first weighs, U.S. Provisional Application 61/138,294 and Swiss Patent application number CH 01979/08 are filed on December 17th, 2008； The entire disclosure of all these applications is incorporated into herein in a manner of clearly quoting.
Technical field
The present invention relates in digital micro-fluid system to the manipulation of droplet size.The art control by and large and Manipulate the liquid of small size, usual micron or nanoscale form.Small liquid volume movement in channel system is known per se , such as controlled by the Micropump in fixing device or the centripetal force in the utensil of rotation test room.However, in digital micro-fluid In system, the voltage of restriction is applied to the electrode of electrod-array so that individual drops are moistened by electricity and are addressed.For electricity The General Introduction of wet method please refers to Washizu, IEEE Transactions on Industry Applications, Volume 34, numbers 4,1998 and Pollack et al., Lab chip, volume 2002,2,96-101.In short, electricity moistening refers to use Microelectrode array (preferably being covered by the dielectric layer including hydrophobic surface) is come the method that moves drop.By to electrode array The selected electrode of row applies the voltage limited, causes the surface tension there are drop on the hydrophobic surface above addressing-electrode Variation.This leads to the significant changes of the liquid-drop contact angle on addressing-electrode, therefore the lateral movement of drop.For this electricity moistening Program, it is known that arrange two kinds of major ways of these electrodes：Drop is caused to move using the single surface with electrod-array Or addition second surface, second surface is opposite with similar electrod-array and at least one grounding electrode is arranged.Electricity moistening skill The major advantage of art is only to need a small amount of liquid, for example, single drop.Therefore it can be executed at liquid in the significantly shorter time Reason.Moreover, the control of liquid movement can lead to the automatic business processing of sample completely under electronic control.
Background technology
The droplet manipulation device of electric moistening is carried out using the single surface (monoplane of electrode is arranged) with electrod-array From known to U.S. Patent number 5,486,337.All electrodes are positioned on the surface of carrier substrate, are transferred in substrate, or by Non-wetable surface covering.Voltage source is connected to electrode.Droplet is set to move by applying voltage to subsequent electrode, therefore Drop square movement number on the electrode is guided according to the voltage application sequence to electrode.Disclose by electrostatic pipette come into Row liquid droplet distribution, electrostatic pipette include the non-wetable pipe of the wettable main electrodes of adjacent tubular, and tubulose is wettable main Electrode is exposed to sample liquids source.The non-wetable secondary electrode of annular is along non-wetable pipe and main electrodes between axial direction It separates, by capillarity, sample liquids enter in the non-wetable secondary electrode of annular.Exist along non-wetable pipe Sequentially apply voltage between main electrodes and secondary electrode.Therefore, a part for sample liquids by main electrodes electrostatic charging simultaneously And then attracted in the form of charged drop by continuous secondary electrode.
Also from the known equipment with the single-sided electrode design for manipulating drop of 6,911,132 B2 of US, all conductions Element is contained on first surface, manipulates drop on the first surface.Additional surface can be parallel to first surface and be arranged to hold Receive drop to be manipulated.Equipment allows droplet manipulation, two droplet coalescences and will such as mix, drop is divided into two Or more drop, continuous liquid flowing is sampled by forming the drop individually controlled from flowing, and to drop Binary or number mixing are iterated to obtain desirable mixed proportion.It is executed using the driving electrodes of at least three alignments Merge, the driving electrodes of three alignments are initially cut off, and first electrode and third electrode are respectively held on covering its surface crown Carrier fluid drips.Continuously, all three electrodes are activated, to aspirate two drops toward each other on second (center) electrode Until they form the single meniscus of two drops of joint.Then, make two external electrodes back to ground state and merge Drop concentrated on the contre electrode still enabled.In order to split the drop of merging again, before enabling all three electrodes, All three electrodes are made to be grounded, to laterally outwardly aspirate the drop of merging and be allowed to propagate on three electrodes.Therefore, Meniscus is shunk to be formed on contre electrode and and by enabling two external electrodes, merge drop and be finally divided into positioned at first Two drops being essentially equal on electrode and third electrode.Merging drop is also disclosed by Washizu 1998.Drop Merge and splits also by Pollack et al. in 2002 disclosures.
Drop is moved using the electrod-array of the apparent surface at least one grounding electrode and carries out micron order control Electric moistening device it is known from US 6,565,727 biplane of electrode (arrange).Each surface of this device may include more A electrode.The driving electrodes of electrod-array are preferably arranged to each other by the protruding portion positioned at each single electrode edge False relation (referring also to 6,911,132 B2 of US).Two facing arrays form gap.It is directed to the table of the electrod-array in gap Face is preferably electrically insulated hydrophobic layer covering.Drop is positioned in gap and by being positioned on the opposite sites in the gap Multiple electrodes be applied continuously in multiple electric fields and moved in monopole filler stream body.It is limited by the hydrophobic surface of control liner Larger area drop meter via small extension be connected to driving electrodes path or driving road, first is off electrode simultaneously And second is coordination electrode.Moistening current potential is firstly applied to pick-off electrode, therefore the liquid on the surface of covering control liner exists It is propagated in pick-off electrode and driving electrodes.Therefore, moistening current potential is removed from pick-off electrode so that pick-off electrode becomes hydrophobic again Property.The partial movement of liquid return to it is contact pad designed, and by nonpolarity filling fluid replace.Therefore, the drop of isolation is divided It opens and is formed in coordination electrode.
The use of this electric moistening device of drop is manipulated in the case of handling biological sample from U.S. Patent Application No. Known to 2007/0217956 Al.Suggest herein for example through thermal cycle amplification of nucleic acid on a printed circuit.By referring to Apply potential between electrode and one or more driving electrodes and transport droplet on an array.Sample is positioned over printed circuit In reservoir on plate, and droplet distributes number on a printed circuit.
It is used to manipulate the container of the sample in drop on thin polymer film from WO 2010/ with thin polymer film Known to 069977 Al.Thin polymer film is positioned on droplet manipulation instrument, and droplet manipulation instrument includes electrod-array and keeps With a certain distance from container bottom side, therefore gap is formed, the manipulation to sample in drop occurs in gap.By being applied to then The drive voltage pulses of driving electrodes execute the separation of drop and the liquid portion for covering several electrodes, and the drop of separation is remote Chaotropic body portion guides.
It is in the shape of integrated structure from a kind of hybrid digital and channel microfluidic device known to 2010/040227 Al of WO Formula, wherein drop can be transported by digital micro-fluid array and be transferred to microfluidic channel.It also discloses by making two lists Only drop is detached at two to be moved on driving road and then combines these electrode paths to merge two individual drops.
It discloses and a kind of is divided by using the dielectric layer being positioned on electrod-array in 2011/002957 A2 of WO Electrode configuration with drop, electrod-array have the part of differing dielectric constant.Wherein about the liquid in digital micro-fluid system Drop manipulates, it is noted that the dielectric constant (k) at certain electrodes is higher, executes the electricity moistening electricity needed for droplet manipulation Pressure is lower, and on the contrary, the dielectric constant (k) at certain electrodes is lower, the electricity moistening voltage needed for execution droplet manipulation is just It is higher.According to this basic principle, " higher " voltage is applied to reservoir electrode and distribution electrode to be moistened using sample liquids Two electrodes.Voltage, which is reduced to " relatively low " value, in region between reservoir electrode and distribution electrode will detach in distribution electricity A part for liquid on extremely.Because the thickness of dielectric constant and selected materials is inversely proportional, change the thickness in dielectric layer region It can be used for limiting the part with differing dielectric constant.Also disclose the low k and high-k dielectric section of combination equal thickness. In addition, also disclosing that the structural obstructions of needs " higher " voltage and the combination with low k and high-k dielectric region.
Goal of the invention and summary
The first object of the present invention is to propose a kind of replacement device, allows to simplify the behaviour to micromation form drop It is vertical.According in a first aspect, realizing this mesh according to first aspect by described herein and disclosed droplet manipulation instrument 's.
The second object of the present invention is to propose a kind of device, allow complete in a manner of simple, automatic and quick Disposition biological sample is integrated, disposition starts from the sample to device provided for analyzing sample biomaterial and ends to realize The processing of final analysis.According to second aspect, realized by biological sample processing system described herein and disclosed According to this purpose of second aspect.
The third object of the present invention is to propose to handle replacing for biological sample using digital micro-fluid sample processing system For method.Using as described herein and disclosed biological sample processing system basis is realized come the method for handling biological sample This purpose of the third aspect.
Additional preferred feature according to the present invention is obtained from appended claims.Advantages of the present invention includes：
The system provides a kind of multi-part device, is adjusted for full automatic treatment biological sample until analysis.
This fully integrated system can directly receive bulk sample (for example, in liquid form or in such as mouth On the surface of solids of chamber swab) and handled using nanoscale volume；It is all these all interactive without using person.
Disposably difference between non-disposable component allows to standardize and with coming in a manner of cost-benefit Carry out automatic business processing.
The method have the characteristics that smaller equal portions reagent can manipulate volume since larger.This permission is larger, arbitrary Fluid Volume introduces box (by user or instrument) without high-precision.This also allows device to be made by large range of user With, including the user of precise volumes cannot be added, and/or make more cheap.
Brief description
To the present invention be explained in greater detail with schematic diagram accoding to exemplary embodiment.However, these explanations will be unlimited The scope of the present invention processed.Moreover, the relative size being shown in the accompanying drawings can be dramatically different because these elements may not according to than Example is drawn.It is shown in the accompanying drawings：
Fig. 1 is the schematic cross-section and section layout of biological sample processing system according to the first aspect of the invention；
Fig. 2 is the top view of the container and film of biological sample processing system according to the present invention, wherein：
Fig. 2A shows at least one well shape structure for the biological sample that the outer edge for being located towards container is arranged；With And
Fig. 2 B are shown for being positioned to be arranged at least one well shape structure of the biological sample in container center；
Fig. 3 is the top view of the different embodiments of preferred electrode array, wherein：
The each electrodes of Fig. 3 A are embodied as rectangular in form；
The each electrodes of Fig. 3 B are embodied as form of hexagons；
The each electrodes of Fig. 3 C are embodied as circular form；And
The each electrodes of Fig. 3 D are embodied as triangular form.
Fig. 4 is according to first preferred embodiment and similar to the grid electrode battle array of arrays of Fig. 3 A with rectangular electrode The top view of row, wherein：
Fig. 4 A：About 18 pulsed drive electrodes of large volume of liquid portion covering identical electrodes array；
Fig. 4 B：It is provided ground voltage pulse with one group of two electrode identical with prior pulse formula driving electrodes；
Fig. 4 C：It is provided ground voltage pulse with one group of three electrode identical with prior pulse formula driving electrodes；
Fig. 5 is the grid electrode of the array with rectangular electrode according to the second preferred embodiment and similar to Fig. 3 A The top view of array, wherein：
Fig. 5 A：About 6 pulsed drive electrodes of large volume of liquid portion covering identical electrodes array；
The additional driving electrodes of Fig. 5 B identical electrodes arrays are activated so that liquid portion now covers about 7 driving electricity Pole；
Fig. 5 C：The direction of additional driving electrodes is enabled with Fig. 5 B on the contrary, enabling another driving electrodes and and prior pulse Identical one group of two electrode of formula driving electrodes are provided ground voltage pulse；
Fig. 6 is the top view according to third preferred embodiment and grid electrode array similar with the array of Fig. 3 A, Its with rectangular electrode and the electrode path adjacent with the array, wherein：
Fig. 6 A：About 10 pulsed drive electrodes of large volume of liquid portion covering electrod-array；
Fig. 6 B：The first two adjacent driven electrode of electrode path is activated, and disables 12 electrodes previously enabled；
Fig. 6 C：The third driving electrodes of electrode path are enabled, disable the first two adjacent driven electrode, and enable electrode array One group of 9 electrode of row, remaining liquid portion now cover about 9 electrodes of electrod-array；
Fig. 6 D：The 4th driving electrodes of electrode path are enabled, disable the second driving electrodes and third driving electrodes, and open With the first driving electrodes of electrode path and adjacent 9 electrodes of electrod-array, three electrodes of electrod-array are disabled；
Fig. 6 E：The 5th driving electrodes of electrode path are enabled, disable the 4th driving electrodes, and before enabling electrode path Three driving electrodes disable all electrodes of electrod-array；
Fig. 6 F：It enables the 5th driving electrodes of electrode path and disables the 4th driving electrodes, enable the of electrode path 6 electrodes of three driving electrodes and the array disable the first two driving electrodes of electrode path and 6 electricity of the array Pole, remaining liquid portion now cover about 8 electrodes of electrod-array.
Specific implementation mode
Fig. 1 shows schematic cross-section and the portion of Exemplary biological samples processing system 1 according to the first aspect of the invention Divide layout.In order to allow to automate and with biological sample is handled in a manner of cost-benefit, this system 1 includes can To be assembled into the different separate parts of a unit (system 1) with simple step.This component may, for example, be container 2, Container 2 includes by biological sample processing system 1.Container 2, which realizes, handles large volume of liquid 18.In the situation of the present invention Under, large volume of liquid is understood as to refer the liquid volume that up to 5ml is up to 10ml, depending on sample to be disposed. For example, using buccal swab, large volume of well shape structure is preferably designed to keep the up to body of 2ml Product；If keeping such as whole blood, well shape structure preferably remains up to 5ml.Container 2 has top side 2 and bottom side 4.In its bottom side 4, container 2 includes protrusion 5.These protrusions 5 can be implemented as the part of the container extended downwardly in bottom side 42.Alternatively, these Protrusion 5 can be individually attached on the bottom side 4 of container 2, such as by glued, welding or other appropriate means these to be dashed forward 5 are played steadily to be attached on the bottom side 4 of container 2.Container 2 includes at least one well shape structure 6.This at least one well shape structure 6 It is opened wide in its top side 7.Therefore, biological sample 9, reaction reagent 10 or the two can be positioned in this well shape structure 6.At it Bottom side 8, at least one well shape structure 6 have at least one opening 11.Channel 12 and container bottom side 4 of this opening by container 2 2 aperture 13 of container connection.It is positioned at least one well shape structure 6 (with or without reaction in liquid 18 or drop 19 Reagent, and/or at least partly with or without biological sample 9) in the case of, it can be by channel 12 from well shape structure 6 are transferred out.The diameter in channel 12 be preferably selected such that capillary force prevent liquid leaked out from aperture 13 and Therefore liquid is retained in 6 inside of at least one well shape structure, without valve or any other closure member in channel 12.Channel 12 Diameter preferably arrives 1mm at 100 μm.
Moreover, biological sample processing system 1 includes flat thin polymer film 14.This flat thin polymer film 14 can also Referred to as " plastic skin ", such as by Yang et al. (2008) on San Diego, the MicroTAS meeting meetings of CA " Exchangeable, pre-loaded " Skin Depot " for digital microfluidics " is proposed.This is flat poly- Closing object film 14 preferably has lower surface 15 and hydrophobic top surface 16.It, can be with as the material for thin polymer film Use food wrapper and stretchable wax film.When in the first step will be single by container 2 is positioned on film 14 When a component is assembled into biological sample processing system 1, the protrusion 5 of container 2 abuts the hydrophobic top surface 16 of film 14.By This, protrusion 5 keeps flat thin polymer film 6 with a certain distance from the bottom side of container 24 at " d ".This distance " d " is by container 2 The height of protrusion 5 is arranged and limits at least one gap 17 when container 2 is positioned on flat thin polymer film 14.In film Gap 17 between 14 upper hydrophobic surface 16 and the bottom side 4 of container is sized to accommodate drop.Preferably, this gap 17 Less than 2mm.Most preferably, gap 17 is less than 1mm.
Biological sample 9 is preferably received in well shape structure 6.It can be mixed with such as buffer solution of liquid 18, be had Or do not have lytic reagent.Biological sample 9 can pass through container 2 from least one well shape structure 6 (while still keeping drop 19) Channel 12 be displaced on the hydrophobic top surface 16 of flat thin polymer film 14.Therefore by the drop 19 with biological sample 9 It is positioned in the gap 17 between film 14 and container 2.
It can prevent liquid from being leaked out from well shape structure 6,6' by applying pressure, centrifugal force or electricity moistening confrontation Capillary force executes displacement, without using valve.It is also possible, however, to use other means, are suitable for liquid 8 or drop 19 It is displaced on the hydrophobic top surface 16 of flat film 14 from well shape structure 6.These displacement means can be used for be stored in The reaction reagent 10 of the well shape structure 6' of container 2 is transferred on the upper surface of film.When by liquid from well shape structure 6,6' shift When on to upper polymeric film surface 16, can 6' for example be discharged via empty well shape structure in the excess air from gap.
In order to manipulate the liquid being preferably displaced to from least one well shape structure 6 of container 2 on the upper surface 16 of film 14 Drop 19, biological sample processing system 1 further includes droplet manipulation instrument 20.This instrument 20 includes：At least one electrod-array 21； Substrate 22 supports at least one electrod-array 21；And control unit 23.Droplet manipulation instrument 20 is implemented such that appearance Device 2 and film 14 are reversibly attached on instrument 20.The lower surface 15 of film 14 abuts electrod-array 21 as a result,.When with this When mode assembles, it is flat poly- that biological sample processing system 1 allows drop 19 to be displaced to from least one well shape structure 16 of container 2 It closes on the upper surface 16 of object film 14 and therefore above at least one electrod-array 21.Electrod-array 21 is implemented as causing Drop 19 moves.Therefore, instrument 20 is implemented as controlling the drop 19 in flat thin polymer film 14 by electricity moistening On upper surface 16 guided movement and there handle biological sample 9.
The canonical biometric sample 9 that can be handled by biological sample processing system 1 is nucleic acid or protein.Preferably, nucleic acid For handling.These nucleic acid include single-stranded or double-stranded DNA (DNA, such as genomic DNA, cDNA, mtDNA), RNA (ribonucleic acid, such as mRNA) and its derivative (for example, nucleic acid of handmarking).These biological samples 9 can hold Such as mouth mucosa cells or hair follicle are contained in tissue sample.Equally, biological sample 9 can be contained in liquid, such as body fluid sample In product, blood, urine, saliva etc..Relevant biological samples 9 can by biological sample processing system 1 according to the present invention into Row processing, it is unrelated with its source.Especially relevant is for example to be derived from patient's (in routine diagnostic procedures) or be derived from crime to show The sample of field (in medical jurisprudence).However, in order to be successfully processed these samples, should be taken based on the material including biological sample 9 The selection of required reaction reagent 10.Purified biological sample 9 can be also loaded at least one well shape structure 6 of container 2 It is interior.In the case, purification step is required during processing not in biological sample processing system 1.
Preferably, at least one well shape structure 6 of container 2 is sized to accommodate solid substrate 24, and solid substrate 24 is held Carry biological sample 9.This solid substrate 24 can be tissue sample.However, it is also possible to which this solid substrate 24 is swab, pressure tongue Plate, needle, syringe, paper such as FTA paper or textile material such as cloth are suitable for carrying and/or collecting biological sample 9 Other substrates such as include the tissue of sample 9.Most preferably, solid substrate 24 is swab head, and therefore, container 2 At least one well shape structure 6 be sized to accommodate swab head.The well shape structure 6 for accommodating swab head is shown in FIG. 1 Exemplary embodiment.The height of diameter and about 40mm of the typical sizes of this well shape structure 6 for swab head with about 10mm Degree.This swab head can for example be made of cotton or polyester, as generally known in the art.These solid samples 24 can To carry tissue sample or in the biological sample 9 (such as body fluid) of liquid form.
In current Fig. 1, container 2 is shown to include the well shape structure 6 and size for being sized to accommodate swab head Different from other well shape structure 6' of sample well shape structure 6.In another modification, as shown in Fig. 2A and Fig. 2 B, container 2 wraps Include at least one sample well shape structure 6 and six smaller well shape structure 6' for storing reaction reagent.These well shape structures 6' preferably sizes are suitable for storage reaction reagent 10 and other required liquid such as buffers.For storing reaction reagent 10 The height of diameter and about 40mm of the typical sizes of this well shape structure 6' with about 5mm.However, it is possible to according to be solved latent The size of each well shape structure 6,6' of container 2 is individually taken in the requirement that problem provides.It is also possible to be grasped according to drop Design, the production method etc. of vertical instrument 20 take the position of the well shape structure 6,6' in container 2.Preferably, well shape structure 6, 6' is positioned in the perimeter of container 2, to provide below container for moving drop 19 and for handling in drop 19 The middle section of sample 9.In order to be handled, the drop of reaction reagent 10 can be transferred in the hydrophobicity of flat thin polymer film 14 It is mixed on surface 16 and there with drop 19.
At least one well shape structure 6 of solid substrate 24 with carrying biological sample 9 can also include reaction reagent 10. Preferably, this reaction reagent 10 is suitable for discharging biological sample 9 from comprising its material.Lytic reagent will be particularly suitable for these Purpose.It may include reaction buffer and opens the device for the cell envelope for keeping biological sample 9 for enzymatic.Reaction reagent 10 can be positioned in well shape structure 6 in liquid form.However, depending on concrete application and availability, reaction reagent can replace It is positioned in well shape structure 6 to generation, such as with lyophilized form.The form of this reaction reagent 10 is anti-with what is preloaded in purchase It is preferred in the case of the container for answering reagent 10.However, the common sense of those skilled in the art think only when freeze-drying process for The reaction reagent of lyophilized form is preferred when the function of reagent 10 is without influencing or being influenced with very little.
In a preferred embodiment, the container 2 of biological sample processing system 1 includes at least one well shape structure 6, well shape knot Structure 6 is implemented as storage reaction reagent 10.Included the feelings for positioning biological sample 9 well shape structure 6 in container 2 Under condition, this embodiment is particularly preferred.Therefore, in this case, container include at least two well shape structures 6, one Well shape structure 6 is for positioning biological sample 9 and a well shape structure 6' for storing reaction reagent.The reagent 10 of storage includes Selected from following reagent：For executing the reagent of cell cracking, the reagent for executing nucleic acid purification, for executing nucleic acid amplification Reagent and reagent for executing nucleic acid sequencing.
During cell cracking, cell integrity is destroyed by opening cell membrane.It can be for example using enzymatic activity Or chemical cracking executes the step.However, the other programs for destroying cell integrity may be suitable.It is exemplary Ground, by ZyGemTMCorporation(Waikato Inno-vation Park,Ruakura Road,Hamilton,New Zealand) the thermostable protease EA1 manufactured should be mentioned herein as the suitable enzymes for executing cell cracking.Alternatively, make Cell cracking is executed with Proteinase K or chemical cracking, both programs are all as known in the art.Matching enzyme used Buffer can be selected by those skilled in the art, without making special effort and being considered as being based on this field Common knowledge.It is well known to those skilled in the art due to executing the program that cell understands, will not be unfolded herein in detail Description.
DNA purification process is also well known to those skilled in the art and will not explain single program step herein. In the case that sample mixture includes the element that may upset following reaction, purification step is preferred.In the situation of the application Under, preferably after cell cracking or after amplification process such as PCR or by synthesis order-checking Need purification step.Preferably, DNA will be purified.It is commonly used for executing the reagent of nucleic acid purification including the pearl being finally modified or grain Son can directly or indirectly combine DNA.After DNA combinations, the improper content of sample mixture can be washed off, and Dissolving DNA in desirable liquid.This pearl can be standard magnetic bead familiar in the field of competence.Advantageous pearl include from The DNA IQ that Promega Corporation (2800Woods Hollow Road, Madison, WI 53711USA) are providedTMOr Person is from Invitrogen Ltd (European Headquarters:3 Fountain Drive,Inchinnan Business Park, Paisley PA4 9RF, UK) provideMagnetic bead.Suitable pearl or particle can also be changed.This modification can To simplify simultaneously stipulated that purifying, and mediate the combination of the DNA of special marking.DNA marker can be realized during amplification program. The exemplary label of primer for PCR is biotin；However, in the present case, can use other Appropriate label.The labeled primer being merged into application can be in subsequent purification process using the coating of such as Streptavidin Pearl captures.However, it is possible to use being suitble to other systems of the DNA of purifying amplification.For example, in this second purification step It can useMagnetic bead.
PCR (PCR) is commonly used in nucleic acid amplification and is also familiar in the field of competence.In short, PCR Include the circulating repetition of three cardinal temperature particular steps：At preferably 98 DEG C detach DNA double-strand denaturation step and The primer (oligonucleotides) of pre-selection is allowed to be attached to the annealing steps of the corresponding sequence on single-stranded, wherein this temperature step depends on In primer sequence, and extend step and be related to polymerase, polymerase is in the combining primer extend of enzyme specific temperature to nucleic acid chains. Polymerase is preferably heat-staple so that it is not exposed to denaturation temperature influence.This heat-stabilised poly familiar in the field of competence Synthase is the polymerase (Taq- polymerases) of thermus aquaticus.It is also possible, however, to use the other thermostabilizations polymerization being commercially available Enzyme.Preferred template is genomic DNA or cDNA.Using PCR, pre-selection, the specific region of template can be expanded, is provided about DNA The more information in source.Wait for that the favored area analyzed by PCR includes mitochondrial DNA (mtDNA), typical short tandem repeat (STR) or become known for for example with the relevant unique single nucleotide polymorphism (SNP) of disease specific.
The sequencing of the DNA of specific amplified is the known tool for further characterizing selected DNA.Mainly sequencing principle is It is familiar in the field of competence, it is sequenced and passes through sequencing by hybridization by expanding.It is related to using the stopping-primer marked by expanding sequencing The relevant processes of PCR, stopping-primer of label randomly terminates elongation process.Then the segment of obtained terminal label Sequence for determining template.It is related to the primer of label being linked to matrix by sequencing by hybridization (SBH).Selection primer makes It is partly overlapped in its sequence.It, can be by analyzing the sequencing that be combined of target after target dna is hybridized to the primer Primer determines sequence.When come when applying sequencing, starting sample treatment by the hybridization step with biological sample processing system 1 Before, the primer of label is preferably linked on the hydrophobic top surface 16 of thin polymer film 14.Most preferably, the primer of label The primer of bookmark before system to be discharged into transaction.
In this case, it is above two or more, preferably all methods should be used according to the present invention Biological sample processing system 1 execute, it includes more than two well shape structure 6,6' to need container 2.Preferably, container 2 includes At least one well shape structure 6 for positioning biological sample 9 and the other well shape structure for storing required reaction reagent 10 6', and a kind of each reaction reagent 10 for method a special well shape structure 6'.Preferably, for positioning biological sample The well shape structure 6 of product 9 is embodied as extraly storing the buffer needed for reaction reagent 10 and cell cracking.Therefore biology is being kept Cell cracking is directly executed in the well shape structure 6 of sample 9.
If executing all methods mentioned above, container 2 includes at least four well shape structures 6,6'：For positioning The well shape structure 6 of biological sample 9 and storage for the reaction reagent 10 of cell cracking；Reaction for stored DNA purifying One well shape structure 6' of reagent 10；A well shape structure 6' for the reaction reagent for storing PCR；And storage is for surveying One well shape structure 6' of the reaction reagent 10 of sequence.Most preferably, container 2 includes at least ten for handling biological sample 9 Well shape structure 6,6'：
At least one well shape structure 6 stores reaction reagent 10 and buffer and for executing for positioning biological sample 9 Cell cracking；
- at least three well shape structure 6' be implemented as purifying before PCR (each for magnetic bead, washing buffer and Elution buffer agent),
- at least two well shape structure 6' for expand (one for storing enzyme and buffer, one for storing primer, Each one primer well shape structure of gene loci to be amplified),
(one for storing Streptavidin coating pearl and one for cleaning after-at least two well shape structure 6' are used for PCR For storing washing buffer),
- at least two well shape structure 6' are for storing reaction reagent 10 and for the buffer (one by sequencing by hybridization For store include reference probe buffer and one storage include unrelated probe buffer).
In general, well shape structure 6, the quantity of 6' depends on the type of reaction system used (required reagent, processing step Suddenly) and the quantity of required analysis (sequence to be analyzed/gene loci quantity, that is, STR, SNP, mtDNA) and can be by ability Field technique personnel are used based on the common knowledge in this field.If the primer for amplification should be stored in container 25, excellent Selection of land, container 2 include for each of amplification program one primer of gene loci to be analyzed.Therefore, 16 genes should analyzed In the case of site, container preferably include 16 for amplification primer well shape structure 6'.In another advantageous variant, amplification Primer needed for step can in a dry form be provided on the hydrophobic top surface 16 of flat thin polymer film 14 so that in order to Primer is stored, individual well shape structure 6' will not needed here in container 2.In the case, using being maintained at well shape knot Buffer in structure 6', primer can be again suspended on film 14.In order to be sequenced by hybridization step, can similarly adopt With the quantity of the well shape structure 6' needed for storage reaction reagent 10 and buffer.
In the especially preferred embodiments, biological sample processing system 1 is used to execute extraction, pure to Relevant biological samples 9 Change, expand and analyzes.Therefore, according in a first aspect, the present invention provides fully integrated system, fully integrated system directly connects By bulk sample (in liquid form or on the surface of solids of such as buccal swab) and using nano-volumes at Reason is analyzed until completing.
In Fig. 2A and Fig. 2 B, container 2 according to the present invention is shown with top view, is had for positioning biological sample 9 A well shape structure 69 and for storing six of reaction reagent 10 other well shape structure 6'.
When directly executing cell cracking in the well shape structure 6 for positioning biological sample 9, discharges and give birth to from cellular context Object sample 9, and be preferably discharged into liquid 18, therefore liquid 18 is the reaction solution obtained from cell cracking.Work as positioning When in the well shape structure 6 of container 2 biological sample 9 and in the case of being not included in one or more cells (if you do not need to Cracking), can be according to following procedure step to select liquid, and it is added to well shape structure 6 via the top side 7 of well shape structure 6 It is interior.In each case, biological sample 9 should be contained at least partly for using biological sample according to the first aspect of the invention In the liquid that product processing system 1 is further processed.Including at least part of liquid 18 or drop of biological sample 9 then from Well shape structure 6 is displaced on the hydrophobic top surface 16 of flat thin polymer film 14 by 2 channel 12 of container to carry out further Processing.
Preferably, by apply pressure, centrifugal force or by electricity moisten, execute displacement, without using valve or it is other can Mobile means.All these preferred displacement means overcome the capillary force action for preventing liquid from being leaked out from well shape structure 6,6'.So And liquid 18 or drop 19 can also be displaced to from well shape structure 6 in the hydrophobicity of flat film 14 using other means On surface 16.
About being further processed, container 2 and flat thin polymer film 14 are reversibly attached on droplet manipulation instrument 20, The lower surface 15 of middle film 14 is against electrod-array 21.Therefore, well shape structure 6 of the drop 19 above electrod-array 21 shifts. In this arrangement, drop 19 can manipulate instrument 20 by liquid in a manner of guided and be shifted by electricity moistening.Control movement To realize the selected processing to the biological sample 9 being contained in the drop 19 and execute this in the preferred sites of electrod-array Kind processing.
In the modification of biological sample processing system 1, drop 19 moves in immiscible liquids system 32 in gap 17. When executing PCR to the biological sample 9 being contained at least one drop 19, this modification is preferred embodiment.It is needed in PCR When this drop 19 is exposed to different temperatures, it is included in about 90 DEG C of denaturing step, it can be by using this unmixing System liquid 32 prevents or at least significantly mitigates liquid evaporation.Not with drop 19 miscible optimum decision system liquid for example selected from Silicone oil, hexadecane and benzene.
It is attached on droplet manipulation instrument 20 to coordinate container 2 in the form of, container 2 and instrument 20 preferably include respectively At least one setting element 25.This setting element is preferably chosen from：
Make at least one of the side zones 28 of container 2 groove and at least one protrusion extended from instrument 20 Groove and protrusion are arranged to form and are cooperated to each other when proper container 2 and film 14 are attached on instrument 20.
At least one groove on the bottom side of container 24 and at least one protrusion extended from machine 20 to work as band When having the container 2 of film 14 to be attached on instrument 20, groove and protrusion, which are arranged to, to be fitted to each other；
At least one groove on the bottom side of container 24 and at least one protrusion extended from instrument 20 to work as band When having the container 2 of film 14 to be attached on instrument 20, groove and protrusion are arranged to form cooperation each other, wherein from instrument 20 At least one protrusion extended is Peltier element locally to provide preselected temperature to container 2；And
Container 2 with irregular polyhedrons shape and the liquid with corresponding groove manipulate instrument so that work as container 2 when being attached to instrument 20, the laminating type alignment that the two coordinates in the form of.
In Fig. 1 (its further groove is on the bottom side of container 2 4), Fig. 2A (side regions of the two of which triangular groove in container In) and Fig. 2 B (side zones of the two of which semi-circular recesses in container 2) in illustrate and be embodied as at least one recessed of container 2 The setting element 25 of slot and at least one protrusion extended from instrument 20.It is heated when using Peltier element for positioning life When the well shape structure 6 of object sample 9, this Peltier element can be implemented as the protrusion extended from instrument 20, can select it Position for example specifically to provide limiting temperature (whether is it in center) to well shape structure 6.It is also possible, however, to use with Limit configuration and container 2 be positioned at other means on droplet manipulation instrument 20, these be it is well known to those skilled in the art simultaneously And it will not be more fully described here.
Although container 2 is positioned on droplet manipulation instrument 20, the drop 19 on flat thin polymer film 14 can only connect It touches the hydrophobic top surface of film 14 or the hydrophobic top surface 16 of film 14 and the bottom side 4 of container 2 can be contacted.This liquid The contact surface of drop 19 may be set (therefore, the size setting of protrusion 5) by the size in gap 19 or 19 size of drop is set Fixing is rung.
Preferably by injection-molded come manufacture container 2.In this way, it is possible to production cost be reduced, although height can be realized Quality is manufactured, and container 2 may be used as the disposable product of low cost.The container 2 of this single use be suitable for sale with For various applications and specific reaction reagent group 10 can be equipped with.Container 2 is preferably by electrically insulating material 26, conductive material 27 Or it is made of the combination of conductive material 26 and electrically insulating material 27.When being made of two kinds of different materials, preferably two steps note Penetrate process.In Fig. 1, Fig. 2A and Fig. 2 B, 2 core of container is made of electrically insulating material 26, wherein around well shape structure 6,6' Region is made of conductive material 27.The peripheral region made of conductive material 27 is separated from each other by insulating materials 26.By conduction material These peripheral regions made of material 27 can form nozzle 47 in the bottom side of container 24, and nozzle 47 is slightly extend into gap 17 (referring to Fig. 1).Drop 19 can differently be generated and drop 19 is delivered in gap by being provided the advantage of by this nozzle, Surface without the bottom side for making the contact container 2 of drop 19.Moreover, this nozzle can allow 19 targeted delivery of drop to gap 17 It is interior.Moreover, the part of peripheral region made of conductive material 27 forms the part in the outside 28 of container 2.This modification can be with It has the following advantages：Each conductive region 27 of container 2 can be individually in electrical contact.This permission is led by voltage control 29 come addressing Electric region and to conductive region provide individual voltage.Therefore, from each well shape structure 6,6', electricity moistening principle can be used to make One or more drops are displaced on the hydrophobic top surface 16 of flat thin polymer film 14.Importantly, each well shape structure It can individually be shifted so that reaction reagent 10 or the liquid containing biological sample 9 can need it on film 14 When individually shift.
In fig. 2, it is arranged towards the outside 28 of container 2 for positioning at least one well shape structure 6 of biological sample 9.Extremely A few well shape structure 6 is extraly surrounded by conductive material 27.In this modification, conductive surrounding 27 extends to form 2 core of container Major part.By this method, the major part of the bottom side 4 of container 2 is also made of conductive material 27.This allows by by container 2 The current-carrying part of bottom side 4 carry out electricity moistening as grounding electrode and handle drop 19, drop 19 is positioned at the hydrophobic of film 14 On property upper surface 16 and contact the bottom side 4 of container 2.Therefore, it in this modification, can further stable droplet 19 be guided Movement.
When container 2 itself undergoes heating stepses, for example, to promote for positioning biological sample and/or reaction reagent When cell cracking at least one well shape structure 6, it is preferable that the part of container 2 is made of thermally insulating material or thermal insulation Gap setting is around higher temperature area.
In a preferred embodiment, container 2 includes the device 30 for mark, for mark device 30 be selected from bar code and RFID (radio frequency identification) is marked or other integrated chips.Due to this identification device 30 be it is well known to those skilled in the art, it Will not be described in more detail here.When the container 2 of biological sample processing system 1 uses in an automated manner, store up simultaneously When depositing the information of the biological sample positioned in the well shape structure 6 about such as container 2, identification device 30 is particularly preferred.This Outside, even specific sample can be tracked in larger laboratory system.
Although the solid substrate 24 including biological sample 9 is positioned at least one well shape structure 6 of container, this well shape Structure 6 preferably includes the maintenance device 31 of the opening 8 for preventing solid substrate 24 from stopping the well shape structure 6.Retaining device Part 31 is selected from filter, frit (referring to Fig. 1) and embossment structure (referring to Fig. 2 B).However, other maintenances familiar in the field of competence Structure 31 can be used for these purposes.
Fig. 2A and Fig. 2 B respectively show the container 2 with analysis area 33.When the hydrophobicity upper table of flat thin polymer film 14 When some regions in face 16 will be close by optical device 38, the container 2 with analysis area 33 is preferred.In simplest implementation In example, cut-out section limiting analysis area 33, preferably in the outside of container 2 28.In the hydrophobicity of the film 14 of suitable order The corresponding region of upper surface 16 can be approached by this method by optical device 38.This optical device can be such as human eye or optics Device.Fig. 2A and Fig. 2 B are schematically illustrated relative to analysis area 33, the optimum position of optical device.Most preferably, it analyzes Area 33 is positioned at the overlying regions of hydrophobic top surface 16, is implemented as handling biological sample 9 using sequencing, logical when executing It is particularly preferred when crossing the sequencing approach of hybridizing method progress.Preferably, Optical devices 38 are selected from standard microscope, photograph Machine system, light guide system such as optical fiber, scanner and its adjustment or combination.For example, in very simple embodiment, camera shooting Machine, simple CCD or PMT (photomultiplier) are used together with light source such as LED, and light source is used as the fluorescence labels on film 14 Excitaton source.If using light guide system, excitation and/or measuring device can be located at 2 side of container.It therefore, can be to phase Automatic sample processing and analysis are executed with thin polymer film 16 and electrod-array 21.
As shown in Figure 2 A and 2 B, when with analysis area 33, container 2 preferably includes side-strut along 45.When with analysis When area 33 abuts, this side-strut extends along 45 along the outside 28 of container 2.Therefore, this side-strut can also be at it along 45 Include one or more protrusions 5 on bottom side, bottom side is attached on the hydrophobic top surface 16 of flat thin polymer film 14.Work as positioning When on film 14, this side-strut is along 45 containers 2 of the bearing with notch.
In such a way that special user is friendly, many containers 2 at least one analysis area 33 are arranged such that often A analysis area 33 is easy to be approached by an Optical devices 38.A kind of possible mode is basic around rotating optics 38 by container 2 Upper circular arrangement.Alternatively, container 2 can be stored with the vertically or horizontally row of suitable carrier, and Optical devices 38 or be had The carrier of 2 row of container is manually or automatically transferred to wherein analysis area 33 by 33 close proximity of Optical devices.
Both container 2 and flat thin polymer film 14 can be used as separate part to be provided to user, and separate part will be protected Hold will just assemble when start to process biological sample 9.However, in alternative embodiments, both parts can be provided as box 40.In the case, box includes container 2 and flat thin polymer film 14, container 2 and flat thin polymer film 14 by glued or Welding or other appropriate means are attached to each other, so that both parts steadily attach.
Preferably, container 2 or box 40 include for protecting well shape structure 6,6' and its content to avoid covering for external action Cover material 43.This covering 43 can be sealingly attached on the top side 7 of container 2.It is attached to be reversible.In advantageous variant In, covering 43 is film, and film is optionally made of pierceable material.In this way, it is possible to the well shape of pre-loaded container 2 Structure 6,6'.Allow safe storage by film cover 43 is applied on container 2.It, will only when starting sample treatment The thorn of film cover 43 is opened and user can be close to the well shape structure 6 of container 2,6'.In addition, container 2 or box 40 can also It is covered by 43 coverings.
In second aspect, the present invention relates to a kind of droplet manipulation instruments 20.In a preferred embodiment, this droplet manipulation instrument Device 20 is implemented as in biological sample processing system 1 according to the first aspect of the invention.However, droplet manipulation instrument 20 It can be used independently of biological sample processing system 1.
Droplet manipulation instrument 20 according to the second aspect of the invention includes for causing drop to move by electricity moistening At least one electrod-array 21.Droplet manipulation instrument 20 further includes the substrate 22 and control unit for supporting electrod-array 21 23.Control unit 23 includes at least one electrode selector 34 being connect at least one voltage control 29.Electrode selector 34 It is implemented as being individually chosen each electrode 35 of electrod-array 21.Moreover, electrode selector 34 is implemented as to selected electrode 35 provide voltage, and voltage is controlled by voltage control 29.At least electrode selector 34 and voltage control 29 is by central processing list Member 36 controls, and central processing unit 36 includes by control unit 23.Central processing unit 36 is implemented as coordination electrode selector 34 and voltage control 29 be individually chosen at least one electrode 35 and to selected electrode 35 provide individual voltage pulse.It is preferred that Ground, individual voltage pulse are selected from ground voltage and driving voltage.By selecting and providing individual voltage pulse, 35 quilt of electrode is selected It is limited to driving electrodes 35' or grounding electrode 35 ".
The electrode 35 of electrod-array 21 can have variously-shaped.In general, be suitable for forming the array of electrode 35 The shape of these electrodes 35 is preferred.Fig. 3 A to Fig. 3 D show certain examples of preferred electrode shape.It can be in terms of Fig. 3 A Go out, electrode 35 there can be rectangular shape.Herein, electrode 35 has square shape, however, other rectangular shapes are also suitable 's.In figure 3b, electrode 35 is shown with hexagon shape, has round in fig. 3 c, and have triangle in fig. 3d Shape shape.However, other shapes are also suitable, as long as electrode 35 can form array and can be approached by electrode contact line.
Preferably, central processing unit 36 includes launchable software 37.This software 27 allows central processing unit 36 Coordination electrode selector 34 and voltage control 29 are to be individually chosen at least one electrode 35 and provide list to selected electrode 35 Only voltage pulse.
Control unit 23 preferably includes power supply 44.This power supply 44 is at least central processing unit 36 and voltage control 29 Electric power is provided.Depending on the embodiment of other elements such as electrode selector 34, power supply 44 can be carried extraly to other elements For electric power.
Control unit 23 can limit the road mobile by guiding of drop 19 by selecting subsequent driving electrodes 35' sequences Diameter.It is at least one then along the path in these selected driving electrodes 35' as a result, under the control of control unit 23 It is provided drive voltage pulses.Moreover, control unit 23 is implemented as essentially simultaneously towards adjacent pulse formula driving electrodes 35' simultaneously And provide ground voltage pulse different from least one electrode 35 " of the selected driving electrodes 35' in the path.
This path mobile by guiding for drop is respectively shown in Fig. 3 A to Fig. 3 D.Indicate subsequent selected drive Moving electrode 35'.Practical driving electrodes 35' is shown as the electrode 35, and drop 19 is located on the electrode 35.Liquid is shown with arrow The guided moving direction of the planning of drop 19.In this direction, driving electricity will be provided along the subsequent electrode 35' in the path Press pulse.
Preferably, the diameter of the size of drop 19 or diameter only slight beyond electrode 35.Most preferably, in order to pass through electricity moistening Moved by guiding, drop not only touches practical driving electrodes 35', but also slightly touches simultaneously and will become next and practical drive The subsequent electrode 35' of moving electrode 35'.However, relative to droplet size adjustment electrode size those skilled in the art knowledge It is interior and will not be repeated here.However, the actual size and design of electrode and the desirable size of drop 19 must be with It is consistent each other and with the convention of electricity moistening.
According to the second inventive aspect, there are at least one grounding electrodes 35 " to be carried to its movement for neighbouring drop 19 to be moved For stablizing effect.Fig. 3 A to Fig. 3 D, which are indicated, can be provided those of ground voltage pulse electrode 35 ".Those grounding electrodes 35 " the selected driving electrodes 35' preferably near pulsed drive electrode 35' and with the path is similar and different.Offer connects Ground voltage pulse is preferably substantially simultaneously executed with offer drive voltage pulses.Alternatively, ground voltage pulse is provided and is carried It is substantially simultaneously carried out for drive voltage pulses.
According to the advantageous variant of droplet manipulation instrument 20, control unit 20 is implemented as to adjacent pulse formula driving electrodes 35' and different from the path selected driving electrodes 35' at least two electrodes 35 " provide ground voltage pulse.Preferably, This at least two selected grounding electrode 35 " is the subsequent electrode 35 on the same side in the path.
As shown in Fig. 3 A to Fig. 3 B, grounding electrode 35 " can be selected from along the path, the neighbouring path and neighbouring drop 19 electrode 35.Preferably, grounding electrode 35 " is selected on the same side in the path.When three or more electrodes 35 " organize base When being provided ground voltage potential simultaneously in sheet, at least two first electrodes 35 " are preferably chosen from the side in the path.However, The remaining electrode 35 " of the group can be chosen such that the side in the path and the first two earthing potential opposite 35 " of the group.So And even if when one group of grounding electrode 35 " is selected from the both sides in the path, they and pulsed drive electrode 35' are substantially simultaneously Or it is provided ground voltage pulse simultaneously.If one group of electrode 35 " is simultaneously provided ground voltage pulse, other electrodes 35 " can With the neighbouring path and in 19 front of drop, the neighbouring path and in 19 rear of drop or both of these case.
In a modification of droplet manipulation instrument 20, one group of 2 or more electrode 35 can be substantially simultaneously provided Drive voltage pulses.In this case, it is possible to which large volume of drop 19' is made to move.However, in this modification, preferably one Group 2 or more electrode 35 substantially simultaneously or is simultaneously provided ground voltage pulse to sufficiently support with large volume of Drop 19'.
In the advantageous variant of droplet manipulation instrument 20, control unit is implemented as stopping at least one selected electrode offer Only voltage pulse stops electrode 35' to generate.Preferably, the stopping voltage pulse providing is different from drive voltage pulses and ground connection Voltage pulse.
For selected electrode 35 to be limited to the voltage pulse of driving electrodes preferably between 20V and 100V.For inciting somebody to action Selected electrode 35 is limited to stop the voltage pulse of electrode preferably between -50V and+50V.Such as the institute in Fig. 3 A to Fig. 3 D Show, select and stop electrode 35 " ' adjacent to the path, it is different from the selected driving electrodes 35' in the path and different from the neighbouring road At least one selected grounding electrode 35 " of diameter.Moreover, stopping electrode 35 " ' these electrodes 35 selected from the neighbouring path, wherein Path provides the direction change that drop 19 moves.Stop electrode 35 " ' support the direction change that is moved along path of drop.
Fig. 3 A to Fig. 3 D show the stopping electrode 35 along the path " ' exemplary possible position.Preferably, along At least one electrode 35 of the path in the position of direction change " ' it is provided stopping voltage pulse.However, it is possible to select at this More than one electrode 35 in region " ' it is provided stopping voltage pulse, as shown in Figure 3D.Herein, in direction change point, two Or more electrode be selected as stop electrode 35 " ' to support drop to move.
Fig. 3 B and Fig. 3 C show the example virtual network of electrod-array 21.Each mesh point 39 of virtual grid is by electricity The geometric center of each electrode 35 of pole array 21 determines.Fig. 3 B show the hexagonal mesh and electrod-array according to hexagon The fine and close packaging of 21 each electrode 35.Fig. 3 C show the orthogonal grid of the quadrature arrangement based on electrode 35, this time base Circular shape is shown on this.Preferably, the subsequent electrode for limiting the path, then selected grounding electrode 35 " and/or with Select afterwards and stop electrode 35 " ' limited by hithermost distance between two mesh points 39 of the virtual grid.By this method, may be used To ensure continuous drop movement.Because the hexagonal arrangement of higher degree of freedom, electrod-array 21 is preferable over quadrature arrangement.
Fig. 1 is schematically shown relative to substrate 22, the position of electrode 35.Preferably, the electrode 35 of electrod-array 21 It is positioned relative to substrate 22 so that the upper surface of electrode 35 and the upper surface of substrate 22 are substantially flush.Alternatively, electrod-array Electrode 35 be positioned in substrate 22 and by substrate closing (referring to left side in Fig. 1).Preferably by electrode 35 as close to The positioning of drop 19 moistens required voltage to reduce electricity.Therefore, the upper surface flush of electrode 35 and substrate 22 and especially It is preferred that very thin thin polymer film.As the material for thin polymer film, food wrapper and stretchable can be used Wax film.
In a preferred embodiment, droplet manipulation instrument 20 according to the present invention is implemented as accommodating and be handled for larger volume Container 2 and at the same time accommodate with hydrophobic top surface 16 flat thin polymer film 14.What is handled for larger volume In the case that container 2 and flat thin polymer film 14 including hydrophobic top surface 16 are attached on droplet manipulation instrument 20, shape The system handled at the biological sample for the sample 9 for being suitable for being positioned in container 2.This system is preferably corresponded to according to this hair The biological sample processing system 1 of bright first aspect.
In another modification, droplet manipulation instrument 20 according to the present invention is implemented as accommodating case 40.The box includes holding Device 2 and flat thin polymer film 14, as herein previously described.The container 2 and film 14 of box 40 pass through gluing Or welding or by steadily connecting other appropriate means of the container 2 with film 14 it is attached to each other.It is attached in this box 40 In the case of on to this modification of droplet manipulation instrument 20, it can be formed at biological sample according to the first aspect of the invention Reason system 1.
In a modification, droplet manipulation instrument 20 includes at least two or more electrod-arrays 21.Preferably, electrode array Row 21 are arranged substantially horizontally in droplet manipulation systems 20.In this modification, instrument 20 is implemented as receiving at least two Or more container 2 and two or more flat thin polymer films 14, or accommodate at least two or more boxes 40.It is excellent Selection of land, container 2 and film 14 or box 40 can be positioned substantially in above electrod-array.However, working as electrod-array not base In sheet horizontal aligument but it is substantially vertical when, these components can be also positioned substantially in side or lateral.
In a particularly preferred embodiment, biological sample processing system 1 according to the first aspect of the invention includes basis The second aspect of the present invention and the droplet manipulation instrument 20 as being hereinbefore discussed in detail.The embodiment of droplet manipulation instrument 20 And the embodiment of biological sample processing system 1 can according to it is to be solved the problem of by selecting various features as discussed above And it selects.If not stated otherwise, the various features provided in this application can be all combined with each other.
In fig. 1 it is shown that including the biological sample processing system 1 of droplet manipulation instrument 20.Droplet manipulation instrument 20 wraps Receiving element 46 is included with safely receiving container 2 and film 14.
In the illustrated embodiment, the setting element 25 of droplet manipulation instrument 20 is included by receiving element 46.
Fig. 3 A show the electrod-array 21 of this particularly preferred embodiment according to biological sample processing system 1.This The amplification vertical view for showing the different sections of Fig. 1, is indicated as the rectangle of dotted line.For the limit relative to electrod-array 21 The position for determining at least one well shape structure 6 of path orientation biological sample 9 is indicated by the empty round wire in Fig. 3 A.At least one The aperture 13 of the access of opening 11, channel 12 at 6 bottom of well shape structure or the bottom side 4 of container 2 is indicated as dotted line respectively Inner circle.Liquid 18 or drop 19 from the channel 12 on the hydrophobic top surface 16 that well shape structure 6 passes through flat thin polymer film 14, It is shifted i.e. above the electrod-array 21 of droplet manipulation instrument 20.Liquid portion 19' is shown at the center of diagram electrod-array 21. This liquid portion 19' covers at least one selected driving electrodes 35' to be isolated with electrode path.Preferably, liquid portion is at least Subsequent electrode 35' and path is partly covered to separate.A according to fig. 3, liquid portion 19' extraly cover electrode 35 ", selection Electrode 35 " is to be provided ground voltage pulse.By being provided to along the subsequent electrode 35' of the initial driving electrodes 35' in the path Drive voltage pulses detach drop 19 and liquid portion 19'.Drop 19 is guided in a first direction then along the path, and And after direction change, drop 19 is guided in a second direction.In direction change position, generate and stop electrode 35 " ' with stabilization Direction change.
After executing cleavage step in the well shape structure 6 of such as container and making liquid portion 19,19' displacements, preferably Ground include the biological sample 6 of well shape structure 6 at least partly, the hydrophobic top surface 16 of flat thin polymer film 14 can be held Row is further processed.In order to handle drop 19 with DNA purification steps, particularly preferably use magnetic bead.In the case, biological sample The droplet manipulation instrument 20 of product processing system 1 preferably includes at least one magnet 41.This magnet 41 is in flat polymer thin Magnetic bead is controlled during being handled in gap 17 on the upper hydrophobicity side 16 of film 14.Suitable magnet can be electromagnet or permanent magnetism Body.Magnet 41 is preferably disposed on the side of instrumented substrate 22, and the side of the substrate 22 of instrument is not by electrod-array 21 Covering.Magnet 41 is alternatively preferably disposed on the side that the substrate 22 of instrument 20 is not abutted with flat thin polymer film 14.
In order to handle the drop 19 for preferably including biological sample 9 using the hot associated process steps of such as PCR, biology The droplet manipulation instrument 20 of sample processing system 1 preferably includes at least one heating element 42.This heating element 42 is preferred Ground is arranged on the side of the substrate 22 of the opposite instrument 20 in the substrate side 14 abutted with flat thin polymer film 14.At least One heating element 42 is implemented as providing on the upper hydrophobic surface 16 of flat thin polymer film 14 to have limiting in advance At least one humidity province of temperature.If droplet manipulation device 20 includes a heating element 42, can be by that will include biology The drop 19 of sample 9 is maintained in single humidity province and executes PCR, while correspondingly changing the temperature in single area.If Using two heating elements 42, can by making to include that the drop 19 of biological sample 9 moves between the areas Liang Ge and to carry out PCR, Temperature wherein needed for circulation step uses the temperature in each area.When handling the liquid for including biological sample 9 by PCR When dripping 19, biological sample processing system 1 includes at least three heating elements 42 in flat polymerization in a particularly preferred modification At least three different temperatures areas are provided on the upper hydrophobic surface 16 of object film 14.Fig. 1 shows biological sample processing system 1, It has three heating elements 42 below supporting substrates 22 and opposite with the side that flat thin polymer film 14 abuts.Often A humidity province has the temperature limited in advance to allow the upper hydrophobic surface 16 to flat thin polymer film 14 to execute PCR.Most Preferably, a humidity province includes the temperature for making double-strandednucleic acid be denaturalized, and a humidity province includes allowing to preselect primer annealing Temperature, and a humidity province includes allowing polymerase that annealing primer is elongated to the temperature of full chain.In addition, at biological sample Reason system 1 may include the 4th heating element 42, and the 4th heating element 42 provides about 4 DEG C of temperature.Using in supporting substrates At least three heating elements 42 of 22 lower sections have the following advantages：Selected temperature may remain in the entire reaction time it is constant and Drop can be moved to another temperature region from a temperature region.This mobile fast temperature allowed in drop 19 becomes Change, than changing the temperature of heating element 42 while keeping drop 19 in place faster.
In another modification of biological sample processing system 1, low vapor pressure liquid layer connects flat thin polymer film 14 The upper surface of lower surface 15 and at least one electrod-array 21 formed with the air bubble for reducing between them.Preferably, low Steam pressure liquid is silicone oil；It is also possible, however, to use using other low vapor pressure liquids.
Fig. 4 shows the grid electrode battle array according to first preferred embodiment and similar to Fig. 3 A with rectangular electrode 35 The top view of row 21.In this partial schematic diagram, particularly preferred droplet manipulation instrument 20, droplet manipulation instrument 20 are shown Including：
(a) at least one electrod-array 21 for being used to cause drop 19 to move by electricity moistening；
(b) substrate 22 of at least one electrod-array 21 is supported；And
(c) control unit 23 comprising at least one electrode selector 34 being connect at least one voltage control 29, until A few electrode selector 34 is implemented as being individually chosen each electrode at least one electrod-array 21 and to selected Electrode 35 provides the voltage controlled by voltage control 29；Control unit 23 further includes being used for coordination electrode selector 34 and voltage control The central processing unit 36 of part 29 is to be individually chosen at least one electrode 35 and provide list at least one selected electrode 35 Only voltage pulse, individual voltage pulse is selected from driving voltage, ground voltage and stops voltage, therefore selected electrode 35 is limited to Driving electrodes 35', grounding electrode 35 " stop electrode 35 " '.
According to the present invention, the control unit 23 of droplet manipulation instrument 20：
It (d) can be by substantially simultaneously selecting one group of the electrod-array 21 two or more subsequent driving electrodes 35' come limit drop 19 and cover electrod-array 21 more than one electrode 35' large volume of liquid portion 19' Guided mobile route, and along the path, select each in driving electrodes 35' to these and driving voltage arteries and veins is provided Punching；And
(e) be implemented as essentially simultaneously towards it is adjacent with pulsed drive electrode 35' or it is identical one group two or more A electrode 35 provides ground connection or stops voltage pulse.
Fig. 4 A show the large volume of liquid for about 18 pulsed drive electrode 35' for covering same electrod-array 21 Body portion 19'.Preferably, these 18 electrodes 35 are activated to collect large volume of these liquid portions 19', the electricity of enabling Pole 35' attracts liquid or at least keeps liquid in stable volume.Most preferably, this large volume of liquid portion 19' is protected It holds on the hydrophobic top surface 16 of flat thin polymer film 14 and in gap 17, flat thin polymer film 14 is to extremely The exposure of substrate 22 (compared to Figure 1 compared with) of a few electrod-array 21.Shown with grey in the electrode 35'(of enabling) around, Multiple electrodes 35 are defined as grounding electrode 35 " or stop electrode 35 " ' (being shown with white).All electrodes 35 operatively connect It is connected to the electrode selector 34 (only partially showing exist but be not shown in Fig. 4 B and Fig. 4 C in Figure 4 A) of control unit 23.
Fig. 4 B are shown is provided ground voltage pulse, one group identical with previous pulsed drive electrode 35' now Two electrodes 35 ".This causes large volume of liquid portion 19' to aspirate and cause larger back towards the electrode 35' of enabling The liquid portion 19' local detachments (referring to double-head arrow) of volume are driven at least two pulseds for still covering same electrod-array 21 Two smaller portions 19' of moving electrode 35'.
Fig. 4 C show identical as prior pulse formula driving electrodes 35' and are provided ground connection now or stop voltage pulse One group of three electrode 35 " of (stop voltage pulse and even promote separating effect).Which results in large volume of liquid portions 19' is now completely separated into at least two pulsed drives electricity that identical electrodes array 21 is still covered (referring to bilateral arrow to the left and right) Two smaller portions 19' of pole 35'.
Similar to the driving path (as shown in Figure 4) for using three row electrodes 35, when the identical particularly preferred droplet manipulation of use The separation (referring to Fig. 5) of smaller size smaller (that is, single drop 19) can also be executed when instrument 20.
Fig. 5 shows the latticed electricity of the array according to the second preferred embodiment and similar to Fig. 3 A with rectangular electrode The top view of pole array.
In fig. 5, about 6 pulsed drive electricity of large volume of liquid portion 19' covering identical electrodes array 21 Pole 35'.Shown with grey in the electrode 35'(of enabling), multiple electrodes 35 are defined as grounding electrode 35 " or stop electrode 35 " ' (being shown with white).All electrodes 35 are operatively connectable to (the only part in fig. 5 of electrode selector 34 of control unit 23 Ground is shown, and is existed but do not showed that in Fig. 5 B and Fig. 5 C).
In order to detach or distribute the drop 19 from large volume of remaining liquid portion 19', identical electrodes array is enabled 21 additional driving electrodes 35' so that liquid portion 19' now covers about 7 driving electrodes 35'(referring to Fig. 5 B, list to the right Side arrow).Then, with the direction that enables additional driving electrodes 35' in Fig. 5 B on the contrary, enabling the additional drive of identical electrodes array 21 Moving electrode 35' makes liquid portion 19', and about 7 driving electrodes 35'(of covering are referring to Fig. 5 B now, unilateral arrow to the right). Then, with the direction that enables additional driving electrodes 35' in Fig. 5 B on the contrary, enable another driving electrodes 35', and and prior pulse Identical one group of two electrode 35 " of formula driving electrodes 35' are provided ground connection or stop voltage pulse.This causes the distribution of drop 19 simultaneously And large volume of remaining liquid portion 19' is moved in different directions (referring to double-head arrow in Fig. 5 C).To one group of two electrode 35 " offer stop pulses even promote desirable separating effect.
When using identical particularly preferred droplet manipulation instrument 20 (referring to Fig. 6), the driving road of three row electrodes 35 is used Diameter (such as shown in Fig. 4) is the same as the combination of the electrode path closest to the single electrode row (compared with Fig. 3 A) for reaching electrod-array 21 It is preferably used for the smaller liquid volume of distribution, that is, single drop 19.
Fig. 6 shows the latticed of the array according to third preferred embodiment and similar to Fig. 3 A with rectangular electrode 35 The top view of the electrode path of electrod-array 21 and neighbouring electrod-array 21.
In fig. 6, about 10 of large volume of liquid portion 19' coverings electrod-array 21 enable electrode 35'.Electricity 35 not enabled of all electrodes (for example, in being grounded or being in stopping potential) in pole path.Preferably, these 12 35 quilts of electrode It enables to collect this large volume of liquid portion 19', the electrode 35' of enabling attracts liquid or at least keeps liquid in stabilization Volume.Most preferably, this large volume of liquid portion 19' is maintained at the hydrophobic top surface of flat thin polymer film 14 On 16 and in gap 17, flat thin polymer film 14 is exposed to the substrate 22 at least one electrod-array 21 (with Fig. 1 Compared to).The electrode for deactivating electrod-array 21 will significantly change the location and shape of large volume of liquid portion 19'.It is all The electrode selector 34 that electrode 35 is operatively connectable to control unit 23 (is only partially shown, in Fig. 6 B and figure in fig. 6 Exist in 6F but do not show that).
In order to which drop 19 is detached or distributed from large volume of remaining liquid portion 19', the first two of electrode path is enabled Adjacent driven electrode 35', and disable 12 previously enabled the electrode 35 of the array 21 (referring to Fig. 6 B).Prohibit in the case With indicate to 12 electrodes 35 of electrod-array 21 provide ground voltage pulse.The first two adjacent driven electrode of electrode path This compound action of 12 electrodes 35 of 35' and the array 21 cause large volume of liquid portion 19' be moved to the left and (referring to unilateral arrow to the left) is propagated on the first two adjacent driven electrode 35' of electrode path.
Continuously (referring to Fig. 6 C), the third driving electrodes 35' of electrode path is enabled, disables the first two adjacent driven electrode 35 ", and also enable one group of 9 electrode 35' of electrod-array.Meanwhile disabling three electrodes 35 " of electrod-array.In this feelings Disabling indicates to provide ground connection or stop pulse to the first two adjacent driven electrode 35 " in the path under condition.This is by making drop 19 It is moved to the third electrode 35 in the path and residual liquid part 19' is made to move backward to the enabling electrode of electrod-array 21 35'(is referring to the unilateral arrow in left and right) and the first drop 19 is caused to be detached with larger volume liquid portion (or distribution).To two phases Adjacent driving electrodes 35 " provide stop pulse and even enhance desirable separating effect.Remaining liquid portion 19' now covers electrode About 9 electrodes of array.
Then (referring to Fig. 6 D), the 4th driving electrodes 35' of electrode path is enabled, the second driving electrodes is disabled and third is driven Moving electrode 35 ".In the case disabling indicate to second driving electrodes and third driving electrodes 35 " in the path provide ground connection or Stop pulse.This causes the first drop 19 to move (referring to unilateral arrow to the left) to the 4th electrode 35' in the path.Meanwhile Together with the first driving electrodes 35' 9 electrode 35' adjacent with electrod-array 21 of electrode path enable and electrod-array three A electrode 35 " keeps disabling.This causes large volume of liquid portion 19' to be moved to the left and first adjacent in electrode path (referring to unilateral arrow to the left) is propagated on driving electrodes 35'.Relative to electrod-array 21 three electrodes 35 " disabling force compared with Electrode 35' concentrations of the residual liquid part 19' of large volume near electrode path.
Hereinafter (referring to Fig. 6 E), enables the 5th driving electrodes of electrode path and disable the 4th driving electrodes.This The first drop 19 is caused to move (referring to unilateral arrow to the left) to the 5th electrode 35' in the path.Meanwhile enabling electrode path First three driving electrodes 35', and disable all electrodes of electrod-array.This cause large volume of liquid portion 19' to It moves left and propagates (referring to unilateral arrow to the left) on first three driving electrodes 35' of electrode path.
Later (referring to Fig. 6 F), the 5th driving electrodes and third driving electrodes 35' of electrode path keep enabling, and the 4th Driving electrodes and the first two driving electrodes disabling.This causes the first drop 19 to be maintained on the 5th electrode 35' and the second drop 19 (referring to unilateral arrow to the left) is detached on the third electrode 35' in the path.Meanwhile 6 electrodes 35 of the array 21 are enabled, This causes remaining liquid portion 19' to be extracted into electrod-array 21 to see, therefrom, now covers about 8 electrodes (referring to list to the right Side arrow).Before other (third) drop 19 of distribution, in each case separated drop 19 moved at least under One driving electrodes 35', and form such as the discribed similar situations of Fig. 6 D.
Particularly, about Fig. 4, Fig. 5 and Fig. 6, it is manifestly intended that preferably, dielectric layer is set to the table of electrod-array 21 On face.Preferably, dielectric layer is the part of the substrate 22 of droplet manipulation instrument 20 or the part of flat thin polymer film 14. Deviate from embodiment shown in Fig. 3 A, gap preferably 17 on its upper side on be closed by the dielectric layer with hydrophobic surface, So that being manipulated in drop 19 or liquid portion 19' movements and the gap 17 between two hydrophobic surfaces.Gap 17 can be with Fluid not miscible with drop 19 or liquid portion 19' is partially or even wholly filled, it is lazy that fluid is preferably chosen from air, chemistry Property gas (such as N2) and non-aqueous liquid (such as silicone oil or hexadecane).Substrate 22 can be selected from such as printed circuit board (PCB), polymerization The materials such as object, glass or combination materials.Preferably, all electrodes 35 are arranged in same level；However, it is also contemplated that by electrode 35 be arranged in Different Plane or different clinoplain in.
In the context of this application, from major part 19' " distribution " drop 19, by drop 19 " being divided into " more droplet, And by major part 19' " being divided into " if stem portion is referred to as manipulating drop 19 or liquid portion 19', no matter larger liquid portion Whether 19', which needs, " is aspirated ".In the context of this application, smaller droplet 19 is made " to combine " each other (for example, being used for hybrid reaction Matching object) or combine with larger liquid portion 19', and " combination " larger liquid portion 19' also referred to as manipulates drop 19 Or liquid portion 19'.In the context of this application, in order to force larger liquid portion 19' desirable direction (for example, To the site of wherein electrode section electrode joint array 21) and control ground and reduce the size of motor array 21 referred to as " suction ". In the context of this application, the quantity of the electrode 35 of equal sizes or " grid " can be referred to as " reservoir " or " manipulate battle array Row ".The spirit and scope of the present invention include belonging in the boundaries of appended claims and to those skilled in the art Any combinations of the different embodiments of rational electrod-array disclosed in the present patent application.
Similar reference numeral refers to similar component, if they are not discussed particularly in detail.
Reference numerals list：
1 biological sample processing system, 2 container
27 conductive material of top side of 3 containers
The outside of 28 container of bottom side of 4 containers
The 29 voltage control of protrusion of 5 containers
6,30 identification device of well shape structure of 6' containers
The 31 maintenance device of top side of 7 well shape structures
32 system liquid of opening of 8 well shape structure bottom sides
9 biological sample, 33 analysis area
10 reaction reagent, 34 electrode selector
35 electrode of opening of 11 well shape structures
The channel 35' driving electrodes of 12 containers
35 " the grounding electrode of aperture of 13 containers
14 flat thin polymer films 35 " ' stop electrode
36 central processing unit of lower surface of 15 flat thin polymer films
37 softwares in the hydrophobicity of 16 flat thin polymer films
38 optical device of surface
17 gap, 39 mesh point
18 liquid, 40 box
19 drop, 41 magnet
Large volume of 42 heating elements of liquid portion of 19'
20 droplet manipulation instrument, 43 covering
21 electrod-array, 44 power supply
The 45 side-strut edge of substrate of 22 instruments
23 control unit, 46 receiving element
24 include 47 nozzle of solid substrate of biological sample
Bottoms of the 25 setting element d in the upper surface of film and container
The distance between 26 electrically insulating material sides

Claims (12)

1. a kind of separation drop (19) and liquid portion in the biological sample processing system (1) including droplet manipulation instrument (20) The method of (19 '), including：
At least one electrod-array (21) has and is used for moistening by causing the drop for covering single electrode (35) by electricity (19) and at least one in the large volume of liquid portion (19 ') of the more than one electrode (35) of the covering electrod-array (21) The electrode (35) of a movement；
Substrate (22) supports at least one electrod-array (21)；
Control unit (23) can limit the guided mobile route of the drop (19) and liquid portion (19 ')；
At least one voltage control (29)；
At least one electrode selector (34) connect described to be individually chosen at least one voltage control (29) Each electrode (35) of at least one electrod-array (21) and individual voltage pulse is provided to the selected electrode (35), it is described It includes driving voltage, ground voltage and the group for stopping voltage that individual voltage pulse, which is selected from,；
Central processing unit (36) is used to control the electrode selector (34) and the voltage control (29)；
Container (2), with top side (3) and bottom side (4), protrusion (5) is distributed on the bottom side (4)；And
Flat thin polymer film (14), with lower surface (15) and hydrophobic top surface (16), the hydrophobic top surface (16) it is kept with a certain distance from the bottom side (4) from the container (2) (d) by the protrusion (5), when the container (2) position In on the flat thin polymer film (14) with its protrusion (5) against flat thin polymer film (14) when, the distance (d) At least one gap (17) is limited,
Wherein it the described method comprises the following steps：
(a) container (2) and the flat thin polymer film (14) are reversibly attached to the droplet manipulation instrument (20) It is upper so that the lower surface (15) of the flat thin polymer film (14) is against at least one electrod-array (21)；
(b) in the gap (17) on the hydrophobic top surface (16) of the flat thin polymer film (14) and Large volume of liquid portion (19 ') is provided above at least one electrod-array (21)；
(c) drive voltage pulses are provided at least one initial driving electrodes (35 ') along the electrode path of restriction, therefore makes institute Large volume of liquid portion (19 ') is stated to move in a first direction；
(d) by providing ground connection or stopping voltage pulse at least one of described initial driving electrodes (35 ') and passing through It is described larger to make that offer drive voltage pulses are organized to two or more driving electrodes (35 ') of the electrod-array (21) The liquid portion (19 ') of volume is moved in a second direction opposite to the first direction to detach the drop (19) and institute State large volume of liquid portion (19 ').
2. according to the method described in claim 1,
It is characterized in that, in step (d), two in the initial driving electrodes (35 ') are provided ground connection or stop voltage arteries and veins Punching is to detach drop (19) and the large volume of liquid portion (19 ').
3. according to the method described in claim 2,
It is characterized in that, in step (d), two in the initial driving electrodes (35 ') are provided stopping voltage pulse to divide Chaotropic drips (19) and the large volume of liquid portion (19 ').
4. according to the method described in claim 1,
It is characterized in that, the container (2) and the flat thin polymer film (14) are arranged to box (40), box (40) packet The container (2) and the flat thin polymer film (14) are included, the container (2) and the flat thin polymer film (14) pass through Gluing is welded and is attached to each other.
5. according to the method described in claim 1,
It is characterized in that, the container (2) and the flat thin polymer film (14) are arranged to start to process remain to i.e. The separate part just assembled when biological sample (9).
6. according to the method described in claim 1, it is characterized in that,
The flat thin polymer film (14) is provided as the flat thin polymer film that is electrically insulated.
7. according to the method described in claim 1,
It is characterized in that, dielectric layer is set on the surface of the electrod-array (21).
8. according to the method described in claim 7,
It is characterized in that, the dielectric layer is the part or described of the substrate (22) of the droplet manipulation instrument (20) The part of flat thin polymer film (14).
9. according to the method described in claim 1,
It is characterized in that, the drop (19) and the large volume of liquid portion (19 ') are in unmixing system liquid (32) It is moved in the inherent gap (17).
10. according to the method described in claim 1,
It is characterized in that, the container (2) is the container (2) handled for larger volume, and it is included in top side (3) and opens To position at least one well shape structure (6) of biological sample (9) and/or reaction reagent (10) wherein；And
Wherein, at least one well shape structure (6) includes bottom side (8), and the bottom side (8) has at least one opening (11), The container (2) further includes channel (12), the channel (12) connect the opening (11) of the well shape structure (6) with it is described Aperture (13) on the bottom side (4) of container (2).
11. according to the method described in claim 10,
It is characterized in that, at least one drop (19) passes through the described of the container (2) from least one well shape structure (6) Channel (12) is displaced on the hydrophobic top surface (16) of the flat thin polymer film (14) and described at least one Above a electrod-array (21), and the electricity moistening controlled by the droplet manipulation instrument (20) is by the flat polymerization Guided movement on the hydrophobic top surface (16) of object film (14) handles the drop (19).
12. according to the method described in claim 1,
It is characterized in that, the container (2) is provided as the disposable product of low cost and by the droplet manipulation instrument The electricity moistening of device (20) control is to the drop (19) on the hydrophobic top surface (16) of the flat thin polymer film (14) It is abandoned after processing.
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