CN105008052A

CN105008052A - Manipulating the size of liquid droplets in digital microfluidics

Info

Publication number: CN105008052A
Application number: CN201380074039.1A
Authority: CN
Inventors: M·N·费格林; J·马莫; M·B·弗兰克林; T·F·李
Original assignee: Tecan Trading AG
Current assignee: Tecan Trading AG
Priority date: 2013-03-04
Filing date: 2013-09-23
Publication date: 2015-10-28
Anticipated expiration: 2033-09-23
Also published as: EP2931425B1; CN105008052B; EP2931425A1; WO2014135232A1

Abstract

A liquid droplet manipulation instrument (20) comprises at least one electrode array (21) for inducing a movement of a liquid droplet (19) by electrowetting; a substrate (22) supporting the at least one electrode array (21); and a control unit (23) comprising at least one electrode selector (34) connected with at least one voltage control (29). The at least one electrode selector (34) is accomplished to individually select each electrode (35) of the at least one electrode array (21) and to provide the selected electrode (35) with a voltage controlled by the voltage control (29). The control unit (23) further comprises a central processing unit (36) for controlling the electrode selector (34) and the voltage control (29) to individually select at least one electrode (35) and to provide the at least one selected electrode (35) with an individual voltage pulse which is selected from a group comprising a drive voltage, a ground voltage, and a stop voltage. The control unit (23) is capable to define a path for a guided movement of a liquid portion (19') of a larger volume that covers more than one electrode (35') of one electrode array (21) by the essentially simultaneous selection of a group of two or more subsequent drive electrodes (35') of said electrode array (21) and to provide each one of these selected drive electrodes (35') with a drive voltage pulse along said path. The control unit (23) is accomplished to essentially simultaneously provide a group of two or more electrodes (35) adjacent to or identical with the pulsed drive electrodes (35') with a ground or stop voltage pulse.

Description

The manipulation of droplet size in digital micro-fluid system

Related application

The application is submission on March 4th, 2013 and is disclosed as US 13/784, the 168 correspondence application of US2013/0175169 Al on July 11st, 2013, and the mode that US 13/784,168 quotes in full is incorporated into herein.US 13/784,168 is the patent application US 13/139 being disclosed as US 2011/0290647 Al, the partial continuous application of 647, patent application US 13/139,647 is American National stage of International Application Serial No. PCT/EP09/67240 of submitting on December 16th, 2009, the claimed U.S. Provisional Application 61/138 of International Application Serial No. PCT/EP09/67240,294 and the priority of Swiss Patent application number CH01979/08, U.S. Provisional Application 61/138,294 and Swiss Patent application number CH01979/08 are filed on December 17th, 2008; The whole open of all these applications is incorporated into herein in the mode of specific reference.

Technical field

The present invention relates to the manipulation to droplet size in digital micro-fluid system.The art controls by and large and handles small size, the liquid of usual micron or nanoscale form.It is that itself is known that little liquid volume in channel system moves, such as, be subject to the Micropump in fixture or the control of the centripetal force in the utensil of rotation test room.But in digital micro-fluid system, the voltage of restriction is applied to the electrode of electrod-array, individual drops is made to pass through electricity moistening and addressed.For the General Introduction of electric wet method, please refer to Washizu, IEEE Transactions on Industry Applications, the 34th volume, numbers 4,1998, and the people such as Pollack, Lab chip, the 2002,2nd volume, 96-101.In brief, electricity moistening finger, uses microelectrode array (dielectric layer being preferably included hydrophobic surface covers) to move the method for drop.By the voltage applying to limit to the selected electrode of electrod-array, cause the surface tension variations hydrophobic surface above addressing-electrode existing drop.This causes the marked change of liquid-drop contact angle on addressing-electrode, being therefore displaced sideways of drop.For the moistening program of this electricity, two kinds of major ways of these electrodes of known arrangement: use the single surface with electrod-array to cause drop to move or to add second surface, second surface is relative with similar electrod-array and arrange at least one earth electrode.The major advantage of the moistening technology of electricity is only to need a small amount of liquid, such as, and single drop.Therefore liquid handling can be performed in the significantly shorter time.And the control of liquid movement completely under Electronic Control, can cause the automatic business processing of sample.

Background technology

Use the single surface (monoplane of electrode is arranged) with electrod-array to carry out the moistening droplet manipulation device of electricity from U.S. Patent number 5,486,337 is known.All electrodes are positioned on the surface of carrier substrate, transfer in substrate, or by non-wetable surface coverage.Voltage source is connected to electrode.By making droplet move to electrode application voltage subsequently, therefore guide the movement number of drop above electrode according to the voltage applying order to electrode.Disclose and carry out liquid droplet distribution by electrostatic pipette, electrostatic pipette comprises the non-wetable pipe of the wettable main electrodes of adjacent tubular, and the wettable main electrodes of tubulose exposes to sample liquid body source.The non-wetable secondary electrode of annular is along non-wetable pipe and main electrodes axially spaced, and by capillarity, sample liquids enters in annular non-wetable secondary electrode.Between main electrodes and secondary electrode, sequentially voltage is applied along non-wetable pipe.Therefore, then a part for sample liquids to be attracted with charged drop form by continuous secondary electrode by main electrodes electrostatic charging.

Also from US 6,911,132 B2 are known has the equipment of the single-sided electrode design for handling drop, and all conducting elements are contained on first surface, handle drop on the first surface.Additional surface can be parallel to first surface and arrange to hold drop to be manipulated.Equipment allows droplet manipulation, such as by two droplet coalescences and mixing, drop is divided into two or more drops, by forming the drop that controls separately from flowing, continuous liquid flowing is sampled, and iteration binary or numeral mixing are carried out to obtain desired mixed proportion to drop.Use at least three drive electrodes aimed to perform merging, three drive electrodes aimed at are cut off at first, and the first electrode and the 3rd electrode respectively carry drop on the surface crown covering it.Continuously, all three electrodes are activated, thus are aspirated toward each other until they form the single meniscus of associatings two drops on second (central authorities) electrode by two drops.Then, two external electrodes are made to turn back to ground state and the drop merged is concentrated on the contre electrode still enabled.In order to again split the drop of merging, before enabling all three electrodes, make all three electrode ground connection, thus outwards aspirate the drop of merging in side direction and make it to propagate on three electrodes.Therefore, shrink meniscus to be formed on contre electrode and and by enabling two external electrodes, to merge drop and being finally divided into the substantially equal drop of two of being positioned on the first electrode and the 3rd electrode.Also disclose merging drop by Washizu 1998.The merging of drop and fractionation are also open 2002 by people such as Pollack.

Use with the apparent surface of at least one earth electrode electrod-array to drop move carry out micron order control electric moistening device from US 6,565,727 biplane of the electrodes (arrange) are known.Each surface of this device can comprise multiple electrode.The drive electrode of electrod-array is preferably arranged to false relation (also referring to US 6,911,132 B2) each other by the protuberance being positioned at each single electrode edge.Two facing arrays form gap.The surface being directed to the electrod-array in gap is preferably electrically insulated hydrophobic layer and covers.Drop to be positioned in gap and by applying multiple electric field continuously to the multiple electrodes on the opposite sites being positioned this gap and moving in one pole filler fluid.Be connected to path or the driving road of drive electrode via little extension by the drop meter controlling the larger area that the hydrophobic surface of liner limits, first be pick-off electrode and second be control electrode.First moistening current potential is applied to pick-off electrode, and therefore the liquid on the surface of Coverage Control liner propagated on pick-off electrode and drive electrode.Therefore, remove moistening current potential from pick-off electrode, make pick-off electrode again become hydrophobic.The part of liquid moves back to contact pad designed, and is replaced by nonpolar fill fluid.Therefore, the drop of isolation is separated and is formed on control electrode.

Known from U.S. Patent Application No. 2007/0217956 Al in the use of this electric moistening device of the situation lower-pilot drop of process biological sample.Here advise such as by thermal cycle amplification of nucleic acid on a printed circuit.Droplet is transported on an array by applying electromotive force between reference electrode and one or more drive electrode.Sample is positioned in the reservoir on printed circuit board (PCB), and the droplet number of distribution on a printed circuit.

Known from WO2010/069977 Al for the container handling the sample in the drop on thin polymer film with thin polymer film.Thin polymer film is positioned on droplet manipulation instrument, and droplet manipulation instrument comprises electrod-array and keeps with a certain distance from container bottom side, therefore forms gap, the manipulation to sample in drop occurs in gap.Perform drop and being separated of liquid part covering some electrodes by the drive voltage pulses being applied to drive electrode subsequently, the drop be separated is guided away from liquid part.

From the known a kind of hybrid digital of WO 2010/040227 Al and passage microfluidic device, it is the form of integrated structure, and wherein, drop can be transported by digital micro-fluid array and transfer to microfluidic channel.Also disclose and merge two individual drops by making two individual drops move and then make these electrode path combine on two separation driving roads.

In WO 2011/002957 A2, disclose a kind of electrode configuration by using the dielectric layer be positioned on electrod-array to distribute drop, electrod-array has the part of differing dielectric constant.Wherein about the droplet manipulation in digital micro-fluid system, it should be noted that, higher at the dielectric constant (k) at some electrode place, the moistening voltage of electricity performed needed for droplet manipulation is lower, and it is contrary, lower at the dielectric constant (k) at some electrode place, the moistening voltage of electricity performed needed for droplet manipulation is higher.According to this general principle, " higher " voltage is applied to reservoir electrode and distributes electrode to utilize moistening two electrodes of sample liquids.In region between reservoir electrode and distribution electrode, voltage is reduced to " lower " value and will be separated in a part for the liquid distributed on electrode.Because the thickness of dielectric constant and selected materials is inversely proportional to, the thickness changing dielectric layer region can be used for limiting the part with differing dielectric constant.Also disclose low k and the high-k dielectric section of combination equal thickness.In addition, the structural obstructions of needs " higher " voltage and the combination with low k and high-k dielectric region is also disclosed.

Goal of the invention and summary

The first object of the present invention is to propose a kind of replacement device, and it allows to simplify the manipulation to microminiaturized form drop.According to first aspect, achieve this object according to first aspect by described herein and disclosed droplet manipulation instrument.

The second object of the present invention is to propose a kind of device, it allows with the fully integrated disposal biological sample of mode simply, automatically and fast, to dispose in the sample auto levelizer that starts from and be provided for analyzing sample biomaterial and to end to achieve the process of final analysis.According to second aspect, realize this object according to second aspect by described herein and disclosed biological sample treatment system.

The third object of the present invention is to propose to use digital micro-fluid sample processing system to process the alternative of biological sample.The method that use processes biological sample with disclosed biological sample treatment system as described herein achieves this object according to the third aspect.

Obtain according to extra preferred feature of the present invention from appended claims.Advantage of the present invention comprises:

This system provides a kind of multi-part device, is adjusted for full automatic treatment biological sample until analyze.

This fully integrated system directly can be received bulk sample (such as, in liquid form or on the surface of solids of such as buccal swab) and be utilized nanoscale volume to process; All these is interactive without the need to user.

Difference between disposable and non-once parts allows with standardization and has cost-benefit mode to carry out automatic business processing.

Feature of the present invention is from larger, to handle less equal portions reagent by volume.This allow comparatively large, arbitrarily Fluid Volume be incorporated into box (by user or instrument) and without the need to high accuracy.This also allows device to be used by user in a big way, comprises the user that can not add precise volumes, and/or makes more cheap.

Brief description

The present invention will be explained in more detail according to exemplary embodiment and schematic diagram.But these are explained and illustrate and will not limit the scope of the invention.And relative size illustrated in the accompanying drawings can be significantly different, because these elements may not proportionally be drawn.Shown in the drawings:

Fig. 1 is schematic cross-section and the section layout of biological sample treatment system according to a first aspect of the invention;

Fig. 2 is according to the container of biological sample treatment system of the present invention and the top view of film, wherein:

Fig. 2 A shows at least one the well shape structure for locating the biological sample arranged towards the outer edge of container; And

Fig. 2 B shows at least one well shape structure that will be arranged in the biological sample of container central authorities for location;

Fig. 3 is the top view of the different embodiments of preferred electrode array, wherein:

The each electrode of Fig. 3 A is embodied as rectangular in form;

The each electrode of Fig. 3 B is embodied as form of hexagons;

The each electrode of Fig. 3 C is embodied as circular form; And

The each electrode of Fig. 3 D is embodied as triangular form.

Fig. 4 is according to the first preferred embodiment and is similar to the top view that Fig. 3 A has the grid electrode array of the array of rectangular electrode, wherein:

Fig. 4 A: the liquid part of larger volume covers about 18 pulsed drive electrodes of identical electrodes array;

Fig. 4 B: there are one group two electrodes identical with prior pulse formula drive electrode and be provided ground voltage pulse;

Fig. 4 C: there are one group three electrodes identical with prior pulse formula drive electrode and be provided ground voltage pulse;

Fig. 5 is according to the second preferred embodiment and is similar to the top view with the grid electrode array of the array of rectangular electrode of Fig. 3 A, wherein:

Fig. 5 A: the liquid part of larger volume covers about 6 pulsed drive electrodes of identical electrodes array;

The extra drive electrode of Fig. 5 B identical electrodes array is activated and makes liquid part now cover about 7 drive electrodes;

Fig. 5 C: contrary with the direction that Fig. 5 B enables extra drive electrode, enables another drive electrode and identical with prior pulse formula drive electrode one group of two electrode is provided ground voltage pulse;

Fig. 6 is the top view of grid electrode array according to the 3rd preferred embodiment and similar to the array of Fig. 3 A, and it has rectangular electrode and the electrode path adjacent with this array, wherein:

Fig. 6 A: about 10 pulsed drive electrodes of the liquid part coated electrode array of larger volume;

Fig. 6 B: the first two adjacent driven electrode of electrode path is activated, and forbid 12 electrodes previously enabled;

Fig. 6 C: the 3rd drive electrode enabling electrode path, forbidding the first two adjacent driven electrode, and enable one group of 9 electrode of electrod-array, remaining fluid body divides about 9 electrodes of existing coated electrode array;

Fig. 6 D: the four-wheel drive electrode enabling electrode path, forbids the second drive electrode and the 3rd drive electrode, and enables the first drive electrode of electrode path and adjacent 9 electrodes of electrod-array, three electrodes of forbidding electrod-array;

Fig. 6 E: the 5th drive electrode enabling electrode path, forbids four-wheel drive electrode, and enables first three drive electrode of electrode path, all electrodes of forbidding electrod-array;

Fig. 6 F: enable the 5th drive electrode of electrode path and forbid four-wheel drive electrode, enable the 3rd drive electrode of electrode path and 6 electrodes of this array, forbidding the first two drive electrode of electrode path and 6 electrodes of this array, remaining fluid body divides about 8 electrodes of existing coated electrode array.

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Detailed description of the invention

Fig. 1 shows schematic cross-section and the section layout figure of Exemplary biological samples treatment system 1 according to a first aspect of the invention.In order to allow with automation and have cost-benefit mode to process biological sample, this system 1 comprises the different separate part that can be assembled into a unit (system 1) with simple step.These parts can be such as containers 2, and container 2 is comprised by biological sample treatment system 1.Container 2 achieves the liquid 18 of process larger volume.In the present case, the liquid of larger volume is understood to relate to nearly 5ml and extremely reaches the liquid volume of 10ml, depends on sample to be disposed.Such as, when using buccal swab, the well shape structure of larger volume is preferably designed to the volume keeping nearly 2ml; If keep such as whole blood, well shape structure is preferably retained to many 5ml.Container 2 has top side 2 and bottom side 4.In its bottom side 4, container 2 comprises projection 5.These projections 5 can be implemented as the part in bottom side 4 to the container 2 of downward-extension.Alternatively, these projections 5 can be attached on the bottom side 4 of container 2 individually, such as, pass through gummed, welding or other suitable means these projections 5 to be stably attached on the bottom side 4 of container 2.Container 2 comprises at least one well shape structure 6.This at least one well shape structure 6 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.In its bottom side 8, at least one well shape structure 6 has at least one opening 11.This opening is connected with container 2 aperture 13 of container bottom side 4 by the passage 12 of container 2.Liquid 18 or drop 19 be positioned over (have or not there is reaction reagent, and/or have or not there is at least part of of biological sample 9) at least one well shape structure 6 when, it can be shifted out from well shape structure 6 by passage 12.The diameter of passage 12 is preferably selected as making capillary force prevent liquid from leaking out from aperture 13 and therefore liquid be retained in inside at least one well shape structure 6, and without the need to other closure member any of valve or passage 12.The diameter of passage 12 preferably arrives 1mm at 100 μm.

And biological sample treatment system 1 comprises smooth thin polymer film 14.This smooth thin polymer film 14 also can be referred to as " plastic skin ", as by the people such as Yang (2008) at San Diego, in the MicroTAS meeting meeting of CA, " Exchangeable, pre-loaded " Skin Depot " fordigital microfluidics " proposes.This smooth thin polymer film 14 preferably has lower surface 15 and hydrophobic top surface 16.As the material for thin polymer film, food wrapper and stretchable wax film can be used.When in a first step by container 2 is positioned on film 14 by single assembling parts to biological sample treatment system 1 time, the projection 5 of container 2 abuts the hydrophobic top surface 16 of film 14.Thus, projection 5 keeps smooth thin polymer film 6 at bottom side 4 " d " place with a certain distance from container 2.This distance " d " is arranged by the height of the projection 5 of container 2 and is limited at least one gap 17 when container 2 is positioned on smooth thin polymer film 14.Gap 17 between the upper hydrophobic surface 16 and the bottom side 4 of container of film 14 is sized to accommodate drop.Preferably, this gap 17 is less than 2mm.Most preferably, gap 17 is less than 1mm.

Biological sample 9 is preferably contained in well shape structure 6.It can mix with liquid 18 such as cushioning liquid, has or does not have lytic reagent.Biological sample 9 can be displaced to by the passage 12 of container 2 hydrophobic top surface 16 of smooth thin polymer film 14 from least one well shape structure 6 (still keeping drop 19) simultaneously.Therefore the drop 19 with biological sample 9 is positioned in the gap 17 between film 14 and container 2.

The capillary force that can prevent liquid from leaking out from well shape structure 6,6' by applying pressure, centrifugal force or electric moistening antagonism performs displacement, without the need to using valve.But also can use other means, it is suitable for liquid 8 or drop 19 to be displaced to the hydrophobic top surface 16 of flat film 14 from well shape structure 6.These displacement means also may be used for the reaction reagent 10 of the well shape structure 6' being stored in container 2 to transfer on the upper surface of film.When being displaced on polymeric film surface 16 from well shape structure 6,6' by liquid, the excess air from gap such as can discharge 6' via the well shape structure of sky.

In order to handle the drop 19 be preferably displaced to from least one well shape structure 6 of container 2 upper surface 16 of film 14, biological sample treatment system 1 also comprises droplet manipulation instrument 20.This instrument 20 comprises: at least one electrod-array 21; Substrate 22, it supports at least one electrod-array 21; And, control unit 23.Droplet manipulation instrument 20 is implemented as and container 2 and film 14 is reversibly attached on instrument 20.Thus, the lower surface 15 of film 14 abuts electrod-array 21.When assembling in this way, biological sample treatment system 1 allows drop 19 to be displaced to the upper surface 16 of smooth thin polymer film 14 and therefore above at least one electrod-array 21 from least one well shape structure 16 of container 2.Electrod-array 21 is implemented as and causes drop 19 to move.Therefore, instrument 20 is implemented as and controls movement that described drop 19 guided on the upper surface 16 of smooth thin polymer film 14 by electricity is moistening and processes biological sample 9 there.

The canonical biometric sample 9 that can be processed by biological sample treatment system 1 is nucleic acid or protein.Preferably, nucleic acid for the treatment of.These nucleic acid comprise DNA (DNA, such as genomic DNA, cDNA, mtDNA), the RNA (ribonucleic acid, such as mRNA) of strand or double-strand, and its derivative (such as, the nucleic acid of handmarking).These biological samples 9 can be contained in such as mouth mucosa cells or hair follicle in tissue sample.Equally, biological sample 9 can be contained in liquid, in such as humoral sample, and such as blood, urine, saliva etc.Relevant biological samples 9 can be processed by biological sample treatment system 1 according to the present invention, has nothing to do with its source.Relevant is especially the sample such as taken from patient's (in routine diagnostic procedures) or take from scene of a crime (in medical jurisprudence).But, in order to successfully process these samples, the selection of required reaction reagent 10 should be taked based on the material comprising biological sample 9.Also purified biological sample 9 can be loaded at least one well shape structure 6 of container 2.In the case, purification step is not required during process in biological sample treatment system 1.

Preferably, at least one well shape structure 6 of container 2 be sized to accommodate solid substrate 24, solid substrate 24 carries biological sample 9.This solid substrate 24 can be tissue sample.But, also may this solid substrate 24 are swabs, spatula, pin, syringe, paper such as FTA paper or textile material such as cloth or be suitable for carry and/or collection of biological sample 9 other substrate or such as comprise the tissue of sample 9.Most preferably, solid substrate 24 is swab heads, and therefore, at least one well shape structure 6 of container 2 be sized to accommodate swab head.Figure 1 illustrates the exemplary embodiment of the well shape structure 6 holding swab head.Typical sizes for this well shape structure 6 of swab head has the diameter of about 10mm and the height of about 40mm.This swab head can be such as made up, as generally known in the art of cotton or polyester.These solid samples 24 can carry tissue sample or the biological sample 9 (such as body fluid) in liquid form.

In current Fig. 1, container 2 is illustrated the well shape structure 6 that comprises and be sized to accommodate swab head and varies in size in other well shape structure 6' of sample well shape structure 6.In another modification, as shown in Fig. 2 A and Fig. 2 B, container 2 comprises at least one sample well shape structure 6 and six less well shape structure 6' for depot reaction reagent.These well shapes structure 6' preferably size is suitable for depot reaction reagent 10 and the liquid such as buffer needed for other.Typical sizes for this well shape structure 6' of depot reaction reagent 10 has the diameter of about 5mm and the height of about 40mm.But the size of each well shape structure 6,6' of container 2 is taked in the requirement that can provide according to potential problems to be solved individually.Equally, the position of the well shape structure 6,6' in container 2 can be taked according to the design of droplet manipulation instrument 20, production method etc.Preferably, well shape structure 6,6' is positioned in the perimeter of container 2, to be provided in below container for mobile drop 19 and the middle section for the treatment of the sample 9 in drop 19.In order to process, the hydrophobic top surface 16 that reaction reagent 10 can transfer to smooth thin polymer film 14 mixes with drop 19 there.

At least one well shape structure 6 with the solid substrate 24 of carrying biological sample 9 also can comprise reaction reagent 10.Preferably, this reaction reagent 10 is suitable for the material release biological sample 9 from comprising it.Lytic reagent will be particularly suitable for these objects.Its device that can comprise reaction buffer and open the cell envelope keeping biological sample 9 for enzymatic.Reaction reagent 10 can be positioned in well shape structure 6 in liquid form.But depend on embody rule and availability, reaction reagent can alternatively be positioned in well shape structure 6, such as, with lyophilized form.The form of this reaction reagent 10 is preferred when buying and having the container of the reaction reagent 10 of prestrain.But the general knowledge of those skilled in the art thinks that only the reaction reagent of lyophilized form is preferred when freeze-drying process does not affect for the function of reagent 10 or have very little impact.

In a preferred embodiment, the container 2 of biological sample treatment system 1 comprises at least one well shape structure 6, and well shape structure 6 is implemented as depot reaction reagent 10.When container 2 comprised for locate biological sample 9 a well shape structure 6, this embodiment is particularly preferred.Therefore, in this case, container comprise at least two well shape structures 6, well shape structure 6 for locate biological sample 9 and a well shape structure 6' for depot reaction reagent.The reagent 10 stored comprises and is selected from following reagent: for perform lysis reagent, for perform nucleic acid purification reagent, for performing the reagent of nucleic acid amplification and the reagent for performing nucleic acid sequencing.

During lysis, destroy cell integrity by opening cell membrane.Can such as use enzymatic activity or chemical cracking to perform this step.But other program destroying cell integrity may be suitable.Exemplarily, by ZyGem ^tMthe thermostable protease EA1 that Corporation (Waikato Inno-vationPark, Ruakura Road, Hamilton, New Zealand) manufactures should here mention as the suitable enzymes for performing lysis.Alternatively, use Proteinase K or chemical cracking to perform lysis, these two kinds of programs are all as known in the art.The buffer mating enzyme used can be selected by those skilled in the art, and without the need to making special effort and by the common practise also thought based on this area.The program understood owing to performing cell is well known to those skilled in the art, and it will here not launch to describe in detail.

DNA purge process is also well known to those skilled in the art and will here explain single program step.When sample mixture comprises the element may upsetting following reaction, purification step is preferred.When the application, preferably after lysis or amplification process such as PCR or by synthesis order-checking after need purification step.Preferably, by purify DNA.Usually, the reagent for performing nucleic acid purification comprises pearl or the particle of final modification, and it can directly or indirectly in conjunction with DNA.DNA combine after, the improper inclusion of sample mixture can be washed off, and in desired liquid dissolving DNA.This pearl can be standard magnetic bead well known in the art.Favourable pearl comprises the DNA IQ provided from Promega Corporation (2800Woods Hollow Road, Madison, WI 53711 USA) ^tMor provide from Invitrogen Ltd (EuropeanHeadquarters:3Fountain Drive, Inchinnan Business Park, Paisley PA49RF, UK) magnetic bead.Also suitable pearl or particle can be revised.This amendment can simplify and specify purifying, and the combination of the DNA of mediation special marking.DNA marker can realize during amplification program.Exemplary label for the primer of PCR is biotin; But, in the present case, other appropriate label can be used.The labeled primer be merged in application can use the pearl of such as Streptavidin coating to catch in purge process subsequently.But, other system of the DNA that applicable purifying can be used to increase.Such as, also can use in this second purification step magnetic bead.

PCR (PCR) is generally used for nucleic acid amplification and is also well known in the art.In brief, PCR comprises being cycled to repeat of three cardinal temperature particular step: the preferably double-strand of 98 DEG C of DNA isolations denaturation step and allow the primer (oligonucleotides) of preliminary election to be attached to the annealing steps of the corresponding sequence on strand, wherein this temperature step depends on primer sequence, and extend step and relate to polymerase, polymerase extends to nucleic acid chains at the combining primer of enzyme specified temp.Polymerase is preferably heat-staple, makes it not be subject to denaturation temperature impact.This heat-stabilised poly synthase well known in the art is the polymerase (Taq-polymerase) of thermus aquaticus.But, also can use other heat-stabilised poly synthase that can buy.Preferred template is genomic DNA or cDNA.Use PCR, can the preliminary election of amplification template, specific region, provide the more information about DNA source.Treat to comprise mitochondrial DNA (mtDNA), typical STR (STR) by the favored area of pcr analysis or become known for such as relevant to disease specific unique SNP (SNP).

What the order-checking of the DNA of specific amplified was used to characterize further selected DNA knows instrument.Main order-checking principle is well known in the art, is checked order and pass through sequencing by hybridization by amplification.The process that the PCR relating to the stopping-primer of usage flag by amplification order-checking is correlated with, the stopping-primer of mark stops elongation process randomly.The fragment of then obtained terminal label is for determining the sequence of template.Related to by sequencing by hybridization (SBH) and the primer strand of mark is received matrix.Primer is selected to make in its sequence partly overlapping.After target dna is hybridized to described primer, the sequencing primer that can be combined by evaluating objects determines sequence.When by applying to check order with the hybridization step of biological sample treatment system 1, before beginning sample treatment, the primer of mark is preferably linked on the hydrophobic top surface 16 of thin polymer film 14.Most preferably, the primer of primer bookmark before system is discharged into transaction of mark.

In this case, above two or more, preferably all methods should use biological sample treatment system 1 according to the present invention to perform, and need container 2 to comprise more than two well shape structures 6,6'.Preferably, container 2 comprises at least one the well shape structure 6 for locating biological sample 9 and the other well shape structure 6' for storing required reaction reagent 10, and a special well shape structure 6' of often kind of reaction reagent 10 for a kind of method.Preferably, the well shape structure 6 for locating biological sample 9 is embodied as depot reaction reagent 10 and the buffer needed for lysis extraly.Therefore in the well shape structure 6 keeping biological sample 9, directly lysis is performed.

If perform all methods mentioned above, so container 2 comprises at least four well shape structures 6,6': for a well shape structure 6 of locating biological sample 9 and store for the reaction reagent 10 of lysis; For a well shape structure 6' of the reaction reagent 10 of stored DNA purifying; For storing a well shape structure 6' of the reaction reagent of PCR; And, store a well shape structure 6' of the reaction reagent 10 for checking order.Most preferably, container 2 comprises at least 10 well shape structures 6,6' for the treatment of biological sample 9:

-at least one well shape structure 6 for locating biological sample 9, depot reaction reagent 10 and buffer and for performing lysis;

-at least three well shape structure 6' are implemented as purifying before PCR (each is for magnetic bead, washing buffer and elution buffer agent),

-at least two well shape structure 6' be used for amplification (one for storing enzyme and buffer, one for storing primer, each gene loci primer well shape structure to be amplified),

Clean after-at least two well shape structure 6' are used for PCR (one for store Streptavidin coating pearl and one for storing washing buffer),

-at least two well shape structure 6' are used for depot reaction reagent 10 and for by the buffer of sequencing by hybridization (one for store the buffer that comprises reference probe and storage comprises the buffer of unrelated probe).

Generally speaking, well shape structure 6, the quantity of 6' depends on the type (required reagent, treatment step) of reaction system used and the quantity (sequence to be analyzed/gene loci quantity of required analysis, that is, STR, SNP, mtDNA) and can be adopted based on the common practise in this area by those skilled in the art.If the primer for increasing should be stored in container 25, preferably, container 2 comprises the gene loci each to be analyzed primer for amplification program.Therefore, when analyzing 16 gene locis, container preferably includes 16 primer well shape structure 6' for increasing.In another advantageous variant, the primer needed for amplification step can be provided in, on the hydrophobic top surface 16 of smooth thin polymer film 14, to make to store primer in a dry form, in container 2, will not need independent well shape structure 6' herein.In the case, use the buffer remained in well shape structure 6', primer can be suspended on film 14 again.In order to be checked order by hybridization step, the quantity of the well shape structure 6' needed for depot reaction reagent 10 and buffer similarly can be adopted.

In the especially preferred embodiments, biological sample treatment system 1 is for performing extraction, purifying, amplification and analysis to Relevant biological samples 9.Therefore, according to first aspect, the invention provides fully integrated system, fully integrated system directly accepts bulk sample (in liquid form or on the surface of solids of such as buccal swab) and utilizes nano-volumes to carry out processing until complete analysis.

In Fig. 2 A and Fig. 2 B, showing according to container 2 of the present invention with top view, there is a well shape structure 69 for locating biological sample 9 and for six of depot reaction reagent 10 other well shape structure 6'.

When directly performing lysis in the well shape structure 6 for locating biological sample 9, from cellular context release biological sample 9, and be preferably discharged in liquid 18, therefore liquid 18 be the reaction solution obtained from lysis.When biological sample 9 is not contained in one or more cell when in the well shape structure 6 being positioned container 2 (if not needing cracking), liquid can be selected according to following program step, and add in well shape structure 6 via the top side 7 of well shape structure 6.In each case, biological sample 9 should be contained in the liquid for using biological sample treatment system 1 to be further processed according to a first aspect of the invention at least in part.Comprise at least part of liquid 18 of biological sample 9 or drop then from well shape structure 6 to be displaced to smooth thin polymer film 14 hydrophobic top surface 16 by container 2 passage 12 to be further processed.

Preferably, moistening by applying pressure, centrifugal force or passing through electricity, perform displacement, without the need to using valve or other movable means.All these preferably the means of being shifted overcome the capillary force action preventing liquid from spilling from well shape structure 6,6'.But, other means can also be used liquid 18 or drop 19 to be displaced to the hydrophobic top surface 16 of flat film 14 from well shape structure 6.

About further process, container 2 and smooth thin polymer film 14 are reversibly attached on droplet manipulation instrument 20, and wherein the lower surface 15 of film 14 is against electrod-array 21.Therefore, the well shape structure 6 of drop 19 above electrod-array 21 is shifted.In this arrangement, drop 19 can handle instrument 20 by electric moistening displacement in the mode guided by liquid.Control mobile to realize the selected process of the biological sample 9 be contained in described drop 19 and to perform this process in the preferred sites of electrod-array.

In the modification of biological sample treatment system 1, drop 19 moves in immiscible liquids system 32 in gap 17.When performing PCR to the biological sample 9 be contained at least one drop 19, this modification is preferred embodiment.When PCR needs this drop 19 to expose to different temperatures, be included in the denaturing step of about 90 DEG C, can prevent or alleviate at least significantly liquid evaporation by using this immiscible system liquid 32.Not miscible with drop 19 optimum decision system liquid is such as selected from silicone oil, hexadecane and benzene.

Be attached on droplet manipulation instrument 20 to be coordinated in the form by container 2, container 2 and instrument 20 preferably respectively comprise at least one setting element 25.This setting element is preferably selected from:

-at least one groove in the side zones 28 of container 2 and make groove and protrusion when container 2 and film 14 are attached to instrument 20 be arranged to form from least one protrusion that instrument 20 extends to be coupled to each other.

-at least one groove on the bottom side 4 of container 2 and making when the container 2 with film 14 is attached to instrument 20 from least one protrusion that machine 20 extends, groove and protrusion are arranged to be fitted to each other;

-at least one groove on the bottom side 4 of container 2 and make when the container 2 with film 14 is attached to instrument 20 from least one protrusion that instrument 20 extends, groove and protrusion are arranged to form each other and coordinate, and at least one protrusion wherein extended from instrument 20 is that Peltier element is to provide preselected temperature partly to container 2; And

-there is the container 2 of irregular polyhedrons shape and there is the liquid manipulation instrument of corresponding groove, make when container 2 is attached to instrument 20, the laminating type that the two coordinates in the form is aimed at.

The setting element 25 of at least one protrusion illustrating at least one groove being embodied as container 2 and extend from instrument 20 in Fig. 1 (its further groove is on the bottom side 4 of container 2), Fig. 2 A (wherein in two triangular grooves side region at container) and Fig. 2 B (wherein two semi-circular recesses are in the side zones of container 2).When using Peltier element to heat the well shape structure 6 for locating biological sample 9, this Peltier element can be implemented as the protrusion extended from instrument 20, and its position can be selected to make such as to provide limiting temperature (no matter whether it is in central authorities) to well shape structure 6 particularly.But also can use to limit other means that container 2 is positioned on droplet manipulation instrument 20 by configuration, these are well known to those skilled in the art and will describe in more detail herein.

Although container 2 is positioned on droplet manipulation instrument 20, the drop 19 on smooth thin polymer film 14 can only contact membrane 14 hydrophobic top surface or can the hydrophobic top surface 16 of contact membrane 14 and the bottom side 4 of container 2.The contact surface of this drop 19 may be subject to size setting (therefore, the size setting of projection 5) or the drop 19 size setting impact in gap 19.

Container 2 is manufactured preferably by injection-molded.In this way, can reduce production cost, although can realize high quality bills of materials, and container 2 can be used as the disposable product of low cost.The container 2 that this single uses is suitable for selling for various application and can being equipped with concrete reaction reagent group 10.Container 2 is preferably by electrically insulating material 26, and conductive material 27 or the combination by conductive material 26 and electrically insulating material 27 are made.When being made up of two kinds of different materials, preferably two step injection process.In Fig. 1, Fig. 2 A and Fig. 2 B, container 2 core is made up of electrically insulating material 26, and the region wherein around well shape structure 6,6' is made up of conductive material 27.The peripheral region be made up of conductive material 27 is separated from each other by insulating materials 26.These peripheral regions be made up of conductive material 27 can form nozzle 47 in the bottom side 4 of container 2, and nozzle 47 extend in gap 17 (referring to Fig. 1) slightly.The advantage provided by this nozzle is differently produce drop 19 and be delivered in gap by drop 19, and does not make the surface of the bottom side of drop 19 contacting container 2.And this nozzle can allow drop 19 targeted delivery in gap 17.And the part of the peripheral region be made up of conductive material 27 forms the part in the outside 28 of container 2.This modification can have the following advantages: each conductive region 27 of container 2 can electrical contact individually.This allows carry out addressing conductive region by voltage control 29 and provide individual voltage to conductive region.Therefore, from each well shape structure 6,6', can make the moistening principle of electricity consumption that one or more drop is displaced on the hydrophobic top surface 16 of smooth thin polymer film 14.Importantly, each well shape structure can be shifted individually, and reaction reagent 10 or the liquid containing biological sample 9 can be shifted individually when needing them on film 14.

In fig. 2, at least one the well shape structure 6 for locating biological sample 9 is arranged towards the outside 28 of container 2.At least one well shape structure 6 is surrounded by conductive material 27 extraly.In this modification, conduction 27 extensions are around with the major part forming container 2 core.In this way, the major part of the bottom side 4 of container 2 is also made up of conductive material 27.This allows to be used as earth electrode by the current-carrying part of the bottom side 4 by container 2 and carries out that electricity is moistening processes drop 19, on the hydrophobic top surface 16 that drop 19 is positioned the film 14 and bottom side 4 of contacting container 2.Therefore, in this modification, can the movement guided of stable droplet 19 further.

When container 2 experience heating steps itself, such as, during to promote for locating the lysis at least one well shape structure 6 of biological sample and/or reaction reagent, preferably, the part of container 2 is made up of hot isolated material or heat insulation gap is arranged at around higher temperature district.

In a preferred embodiment, container 2 comprises the device 30 for identifying, and the device 30 for identifying is selected from bar code and RFID (radio frequency identification) marks or other integrated chip.Because this identification device 30 is well known to those skilled in the art, they will not be described in more detail herein.When the container 2 of biological sample treatment system 1 uses in an automated manner, when storing the information of the biological sample of location in the well shape structure 6 about such as container 2, identification device 30 is particularly preferred simultaneously.In addition, even specific sample can be followed the tracks of in comparatively big experiment chamber system.

Although the solid substrate 24 comprising 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 for preventing solid substrate 24 from stopping the opening 8 of described well shape structure 6.Maintenance device 31 is selected from filter, frit (referring to Fig. 1) and embossment structure (referring to Fig. 2 B).But other retention system 31 well known in the art also may be used for these objects.

Fig. 2 A and Fig. 2 B respectively shows the container 2 with analysis area 33.When the hydrophobic top surface 16 of smooth thin polymer film 14 some region will by optics 38 close to time, the container 2 with analysis area 33 is preferred.In the simplest embodiments, cut-out section limiting analysis district 33, preferably in the outside 28 of container 2.In this way can be close by optics 38 at the respective regions of the hydrophobic top surface 16 of the film 14 of suitable order.This optics can be such as human eye or Optical devices.Fig. 2 A and Fig. 2 B schematically illustrates relative to analysis area 33, the optimum position of optics.Most preferably, analysis area 33 is positioned the overlying regions of hydrophobic top surface 16, and it is implemented as and uses order-checking to process biological sample 9, when performing the sequence measurement undertaken by hybridizing method, is particularly preferred.Preferably, Optical devices 38 be selected from standard microscope, camera arrangement, light guide system such as optical fiber, scanner and its adjust or combine.Such as, in very simple embodiment, video camera, simple CCD or PMT (photomultiplier) uses together with light source such as LED, and light source is used as the excitaton source of the fluorescence labels on film 14.If use light guide system, excitation and/or measurement mechanism can be positioned at container 2 side.Therefore, automatic sample process and analysis can be performed to same polymer film 16 and electrod-array 21.

As shown in Figure 2 A and 2 B, when having analysis area 33, container 2 preferably includes side-strut along 45.When adjoining with analysis area 33, this side-strut extends along 45 along the outside 28 of container 2.Therefore, this side-strut also can comprise one or more projection 5 along 45 on its bottom side, and bottom side is attached on the hydrophobic top surface 16 of smooth thin polymer film 14.When being positioned on film 14, this side-strut has the container 2 of otch along 45 supportings.

In the mode of special user close friend, many containers 2 with at least one analysis area 33 are arranged such that each analysis area 33 is easy to close by Optical devices 38.A kind of may mode by container 2 around rotating optics 38 circular arrangement substantially.Alternatively, container 2 can with suitable carrier vertically or level row store, and Optical devices 38 or the carrier with container 2 row transfer to wherein analysis area 33 manually or automatically by the close position of Optical devices 33.

Both container 2 and smooth thin polymer film 14 can provide to user as separate part, and separate part will remain to when being about to start to process biological sample 9 and just assemble.But in alternative embodiments, these two parts can be provided as box 40.In the case, box comprises container 2 and smooth thin polymer film 14, and container 2 and smooth thin polymer film 14 are by gluing together or welding or other suitable means are attached to each other, to make these two parts stably attached.

Preferably, container 2 or box 40 comprise the covering 43 avoiding external action for the protection of well shape structure 6,6' and its inclusion.This covering 43 can be attached on the top side 7 of container 2 hermetically.Attached can be reversible.In advantageous variant, covering 43 is films, and film is alternatively by can pierceable material make.In this way, can well shape structure 6, the 6' of pre-loaded container 2.Safe storage is allowed by being applied on container 2 by film cover 43.Only when starting sample treatment, film cover 43 thorn is opened and user can close to well shape structure 6, the 6' of container 2.In addition, container 2 or box 40 also can be coated to cover material 43 and cover.

In second aspect, the present invention relates to a kind of droplet manipulation instrument 20.In a preferred embodiment, this droplet manipulation instrument 20 is implemented as in biological sample treatment system 1 according to a first aspect of the invention.But droplet manipulation instrument 20 can use independent of biological sample treatment system 1.

Droplet manipulation instrument 20 according to a second aspect of the invention comprises for by moistening at least one electrod-array 21 causing drop movement of electricity.Droplet manipulation instrument 20 also comprises substrate 22 for support electrode array 21 and control unit 23.Control unit 23 comprises at least one electrode selector 34 be connected with at least one voltage control 29.Electrode selector 34 is implemented as each electrode 35 of choice electrode array 21 individually.And electrode selector 34 is implemented as provides voltage to selected electrode 35, voltage is subject to voltage control 29 and controls.At least electrode selector 34 and voltage control 29 are subject to CPU 36 and control, and CPU 36 is comprised by control unit 23.CPU 36 is implemented as control electrode selector 34 and voltage control 29 to select at least one electrode 35 individually and to provide individual voltage pulse to selected electrode 35.Preferably, individual voltage pulse is selected from ground voltage and driving voltage.By selecting and providing individual voltage pulse, selected electrode 35 is restricted to drive electrode 35' or earth electrode 35 ".

The electrode 35 of electrod-array 21 can have various shape.Generally speaking, the shape being suitable for these electrodes 35 of the array forming electrode 35 is preferred.Fig. 3 A to Fig. 3 D shows some example of preferred electrode shape.Can find out from Fig. 3 A, electrode 35 can have rectangular shape.Herein, electrode 35 has square shape, but other rectangular shape is also suitable.In figure 3b, electrode 35 is shown having hexagon shape, has toroidal in fig. 3 c, and has triangular shaped in fig. 3d.But other shape is also suitable, as long as electrode 35 can form array and can be close by electrode contact line.

Preferably, CPU 36 comprises launchable software 37.This software 27 allows CPU 36 control electrode selector 34 and voltage control 29 to select at least one electrode 35 individually and to provide individual voltage pulse to selected electrode 35.

Control unit 23 preferably includes power supply 44.This power supply 44 is at least CPU 36 and voltage control 29 provide electric power.Depend on the embodiment of other element such as electrode selector 34, power supply 44 can provide electric power to other element extraly.

Control unit 23 limit by selecting drive electrode 35' sequence subsequently drop 19 by the path guiding movement.Thus, under the control of control unit 23, in these selected drive electrode 35', at least one is provided drive voltage pulses along described path subsequently.And control unit 23 is implemented as essentially simultaneously towards adjacent pulse formula drive electrode 35' and is different from least one electrode 35 of the selected drive electrode 35' in this path " ground voltage pulse is provided.

For drop by guiding this path of movement respectively shown in Fig. 3 A to Fig. 3 D.Indicate selected drive electrode 35' subsequently.Actual drive electrode 35' is shown as this electrode 35, and drop 19 is positioned on this electrode 35.The moving direction guided of the planning of drop 19 is shown with arrow.In the direction in which, drive voltage pulses will be provided along this path with rear electrode 35'.

Preferably, the size of drop 19 or diameter are only slight beyond the diameter of electrode 35.Most preferably, in order to be undertaken by guiding mobile by electricity is moistening, drop not only touches actual drive electrode 35', and touch slightly simultaneously by become next actual drive electrode 35' with rear electrode 35'.But, will here not repeat in the knowledge of those skilled in the art relative to droplet size adjustment electrode size.But, the desired size of the actual size of electrode and design and drop 19 must with each other and the convention moistening with electricity conform to.

According to the second inventive aspect, there is at least one earth electrode 35 in contiguous drop 19 to be moved " move to it stablizing effect is provided.Fig. 3 A to Fig. 3 D indicates those electrodes 35 that can be provided ground voltage pulse ".Those earth electrodes 35 " preferably adjacent pulse formula drive electrode 35' and similar and different with the selected drive electrode 35' in this path.There is provided ground voltage pulse preferably substantially to perform with providing drive voltage pulses simultaneously.Alternatively, ground voltage pulse is provided and provides drive voltage pulses substantially to carry out simultaneously.

According to the advantageous variant of droplet manipulation instrument 20, control unit 20 is implemented as to adjacent pulse formula drive electrode 35' and is different from least two electrodes 35 of the selected drive electrode 35' in this path " ground voltage pulse is provided.Preferably, these at least two selected earth electrodes 35 " be in the same side in this path with rear electrode 35.

As shown in Fig. 3 A to Fig. 3 B, earth electrode 35 " can be selected from along this path, be close to this path and the electrode 35 of contiguous drop 19.Preferably, selected earth electrode 35 " in the same side in this path.When three or more electrodes 35 " group is when being substantially provided ground voltage potential simultaneously, at least two the first electrodes 35 " be preferably selected from the side in this path.But, the remaining electrode 35 of this group " can be chosen to make the first two earthing potential relative 35 of the sidepiece in this path and this group ".But, even if when one group of earth electrode 35 " selects from the both sides in this path, they and pulsed drive electrode 35' substantially while or be provided ground voltage pulse simultaneously.If one group of electrode 35 " be simultaneously provided ground voltage pulse, other electrode 35 " this path can be close to and in drop 19 front, this path contiguous and at drop 19 rear or both of these case.

In a modification of droplet manipulation instrument 20, one group 2 or more electrodes 35 can be provided drive voltage pulses substantially simultaneously.In the case, the drop 19' of larger volume can be made to move.But in this modification, preferably one group 2 or more electrodes 35 substantially simultaneously or be provided ground voltage pulse fully to support the drop 19' with larger volume simultaneously.

In the advantageous variant of droplet manipulation instrument 20, control unit is implemented as provides stopping voltage pulse stopping electrode 35' to generate at least one selected electrode.Preferably, the stopping voltage pulse providing is different from drive voltage pulses and ground voltage pulse.

For selected electrode 35 is defined as the voltage pulse of drive electrode preferably between 20V and 100V.Stop the voltage pulse of electrode preferably between-50V and+50V for being defined as by selected electrode 35.As shown in Fig. 3 A to Fig. 3 D, selected stopping electrode 35 " ' be close to this path, be different from the selected drive electrode 35' in this path and be different from least one the selected earth electrode 35 being close to this path ".And, stop electrode 35 " ' be selected from these electrodes 35 being close to this path, wherein path provides the direction of drop 19 movement to change.Stop electrode 35 " ' support that drop changes along the direction of path movement.

Fig. 3 A to Fig. 3 D shows the stopping electrode 35 along this path " ' exemplary possible position.Preferably, at least one electrode 35 of the position changed along this path in direction " ' be provided stopping voltage pulse.But, more than one electrode 35 in this region can be selected " ' be provided stopping voltage pulse, as shown in Figure 3 D.Herein, at direction change point, two or more electrodes are selected as and stop electrode 35 " ' to support that drop moves.

Fig. 3 B and Fig. 3 C shows the example virtual network of electrod-array 21.Each mesh point 39 of virtual grid is determined by the geometric center of each electrode 35 of electrod-array 21.Fig. 3 B shows the densification packaging according to each electrode 35 of hexagonal hexagonal mesh and electrod-array 21.Fig. 3 C shows the orthogonal grid of the quadrature arrangement based on electrode 35, and this time essentially show round-shaped.Preferably, for limit this path with rear electrode, selected earth electrode 35 subsequently " and/or selectedly subsequently stop electrode 35 " ' hithermost distance limits between two mesh points 39 by this virtual grid.In this way, can guarantee that continuous drop moves.Because the higher free degree, the hexagonal arrangement of electrod-array 21 is preferable over quadrature arrangement.

Fig. 1 schematically shows relative to substrate 22, the position of electrode 35.Preferably, the electrode 35 of electrod-array 21 is located relative to substrate 22, and the upper surface of electrode 35 is flushed substantially with the upper surface of substrate 22.Alternatively, the electrode 35 of electrod-array to be positioned in substrate 22 and to be closed in (referring to left side in Fig. 1) by substrate.Preferably electrode 35 is located can reduce the moistening required voltage of electricity near drop 19 as far as possible.Therefore, the upper surface flush of electrode 35 and substrate 22 and particularly preferably very thin thin polymer film.As the material for thin polymer film, food wrapper and stretchable wax film can be used.

In a preferred embodiment, droplet manipulation instrument 20 according to the present invention is implemented as the container 2 that holds for larger volume process and holds simultaneously and has the smooth thin polymer film 14 of hydrophobic top surface 16.When the container 2 for larger volume process is attached on droplet manipulation instrument 20 with the smooth thin polymer film 14 comprising hydrophobic top surface 16, form the system being suitable for the biological sample process of the sample 9 be positioned in container 2.This system preferably corresponds to biological sample treatment system 1 according to a first aspect of the invention.

In another modification, droplet manipulation instrument 20 according to the present invention is implemented as accommodation box 40.Described box comprises container 2 and smooth thin polymer film 14, as in this article previously described.The container 2 of box 40 and film 14 are by gluing together or welding or attached to each other by stably connecting container 2 and other suitable means of film 14.In this modification that this box 40 is attached to droplet manipulation instrument 20, biological sample treatment system 1 according to a first aspect of the invention can be formed.

In a modification, droplet manipulation instrument 20 comprises at least two or more electrod-arrays 21.Preferably, electrod-array 21 is essentially horizontally arranged in droplet manipulation systems 20.In this modification, instrument 20 is implemented as accommodation at least two or more containers 2 and two or more smooth thin polymer films 14, or holds at least two or more boxes 40.Preferably, container 2 and film 14 or box 40 can be positioned above electrod-array substantially.But, when electrod-array not substantial horizontal aim at but substantially vertically time, also these parts can be positioned side or side direction substantially.

In one particularly preferably embodiment, biological sample treatment system 1 according to a first aspect of the invention comprises according to a second aspect of the invention and as the droplet manipulation instrument 20 discussed in detail hereinbefore.The embodiment of droplet manipulation instrument 20 and the embodiment of biological sample treatment system 1 can according to the problem that will solve by selecting various feature and selecting as discussed above.If do not illustrated in addition, the various features provided in this application can whole combinations with one another.

In FIG, the biological sample treatment system 1 comprising droplet manipulation instrument 20 is shown.Droplet manipulation instrument 20 comprises receiving element 46 to receive container 2 and film 14 safely.

In the illustrated embodiment, the setting element 25 of droplet manipulation instrument 20 is comprised by receiving element 46.

Fig. 3 A shows the electrod-array 21 of this particularly preferably embodiment according to biological sample treatment system 1.This diagram illustrates the amplification vertical view of the different portions section of Fig. 1, be indicated as the rectangle of dotted line.Position at least one well shape structure 6 of the restriction path orientation biological sample 9 relative to electrod-array 21 is indicated by the empty round wire in Fig. 3 A.Bottom at least one well shape structure 6, the opening 11 at place, the aperture 13 of the path of passage 12 or the bottom side 4 of container 2 are indicated as dotted line inner circle respectively.Liquid 18 or drop 19, namely shift from well shape structure 6 by the passage 12 hydrophobic top surface 16 of smooth thin polymer film 14 above the electrod-array 21 of droplet manipulation instrument 20.Liquid part 19' illustrates at the center of diagram electrod-array 21.This liquid part 19' covers at least one selected drive electrode 35' and electrode path is isolated.Preferably, the electrode 35' that liquid part covers subsequently at least in part separates with path.According to Fig. 3 A, liquid part 19' is coated electrode 35 extraly ", choice electrode 35 " to be provided ground voltage pulse.Precipitation of liquid droplets 19 and liquid part 19' is come by providing drive voltage pulses to drive electrode 35' initial along this path electrode 35' subsequently.Then guide drop 19 in a first direction along this path, and after the change of direction, guide drop 19 in a second direction.At direction change location, generate and stop electrode 35 " ' change with stabilising direction.

In the well shape structure 6 of such as container, perform cleavage step and make liquid part 19,19' be shifted after, preferably include at least part of of the biological sample 6 of well shape structure 6, further process can be performed to the hydrophobic top surface 16 of smooth thin polymer film 14.In order to DNA purification step process drop 19, particularly preferably use magnetic bead.In the case, the droplet manipulation instrument 20 of biological sample treatment system 1 preferably includes at least one magnet 41.Magnetic bead is controlled during process in the gap 17 of this magnet 41 on the upper hydrophobicity sidepiece 16 of smooth thin polymer film 14.Suitable magnet can be electromagnet or permanent magnet.Magnet 41 is preferably arranged on this sidepiece of instrumented substrate 22, and the sidepiece of the substrate 22 of instrument is not covered by electrod-array 21.On the sidepiece that the substrate 22 that magnet 41 is alternatively preferably arranged in instrument 20 does not abut with smooth thin polymer film 14.

Preferably include the drop 19 of biological sample 9 to utilize the hot associated process steps of such as PCR to process, the droplet manipulation instrument 20 of biological sample treatment system 1 preferably includes at least one heating element heater 42.This heating element heater 42 is preferably arranged on this sidepiece of the substrate 22 of the instrument 20 relative with the substrate sidepiece 14 that smooth thin polymer film 14 abuts.At least one heating element heater 42 is implemented as at least one humidity province providing on the upper hydrophobic surface 16 of smooth thin polymer film 14 and have the temperature limited in advance.If droplet manipulation device 20 comprises a heating element heater 42, by being remained in single humidity province by the drop 19 comprising biological sample 9 and performing PCR, can correspondingly change the temperature in single district simultaneously.If use two heating element heaters 42, can carry out PCR by making the drop 19 comprising biological sample 9 move between Liang Ge district, the temperature wherein needed for circulation step adopts the temperature in each district.When processing by PCR the drop 19 comprising biological sample 9, biological sample treatment system 1 one particularly preferably modification comprise at least three heating element heaters 42 to provide at least three different temperatures districts on the upper hydrophobic surface 16 of smooth thin polymer film 14.Fig. 1 shows biological sample treatment system 1, and it has three heating element heaters 42 below supporting substrates 22 and relative with the sidepiece that smooth thin polymer film 14 abuts.Each humidity province has the temperature limited in advance and performs PCR with permission to the upper hydrophobic surface 16 of smooth thin polymer film 14.Most preferably, humidity province comprises the temperature for making double-strandednucleic acid sex change, and a humidity province comprises the temperature allowing preliminary election primer annealing, and a humidity province comprises the temperature allowing polymerase annealing primer to be elongated to full chain.In addition, biological sample treatment system 1 can comprise the temperature that the 4th heating element heater the 42, four heating element heater 42 provides about 4 DEG C.At least three heating element heaters 42 below supporting substrates 22 are utilized to have the following advantages: selected temperature can remain on that the whole reaction time is constant and drop can move to another temperature province from a temperature province.The fast temperature change of this mobile permission in drop 19, it is faster than the temperature changing heating element 42 while keeping drop 19 in place.

In another modification of biological sample treatment system 1, the lower surface 15 that low vapor pressure liquid layer connects smooth thin polymer film 14 is formed with the air bubble reduced between which with the upper surface of at least one electrod-array 21.Preferably, low vapor pressure liquid is silicone oil; But, also can use other low vapor pressure liquid.

Fig. 4 shows according to the first preferred embodiment and is similar to the top view that Fig. 3 A has the grid electrode array 21 of rectangular electrode 35.In this partial schematic diagram, show particularly preferred droplet manipulation instrument 20, droplet manipulation instrument 20 comprises:

A () is for by moistening at least one electrod-array 21 causing drop 19 movement of electricity;

B () supports the substrate 22 of at least one electrod-array 21; And

(c) control unit 23, it comprises at least one electrode selector 34 be connected with at least one voltage control 29, and at least one electrode selector 34 is implemented as to be selected each electrode at least one electrod-array 21 individually and provides powered voltage-controlled 29 voltage controlled to selected electrode 35; Control unit 23 also comprises CPU 36 for control electrode selector 34 and voltage control 29 to select at least one electrode 35 individually and to provide individual voltage pulse at least one selected electrode 35, individual voltage pulse is selected from driving voltage, ground voltage and stopping voltage, therefore selected electrode 35 is defined as drive electrode 35', earth electrode 35 " or stop electrode 35 " '.

According to the present invention, the control unit 23 of droplet manipulation instrument 20:

(d) by substantially select simultaneously one group of described electrod-array 21 two or more subsequently drive electrode 35' to the mobile route guided of liquid part 19' of larger volume of more than one electrode 35' limiting drop 19 and cover an electrod-array 21, and along described path, in these selected drive electrode 35', each provides drive voltage pulses; And

E () is implemented as provides ground connection essentially simultaneously towards one group of two or more electrode 35 adjacent or identical with pulsed drive electrode 35' or stops voltage pulse.

Fig. 4 A shows the liquid part 19' of the larger volume of about 18 the pulsed drive electrode 35' covering same electrod-array 21.Preferably, these 18 electrodes 35 are activated these liquid part 19' collecting larger volume, and the electrode 35' enabled attracts liquid or at least keeps liquid at stable volume.Most preferably, on the hydrophobic top surface 16 that the liquid part 19' of this larger volume remains on smooth thin polymer film 14 and in gap 17, smooth thin polymer film 14 exposes (compared to Figure 1 comparatively) to the substrate 22 with at least one electrod-array 21.Illustrate with grey at the electrode 35'(enabled) around, multiple electrode 35 is restricted to earth electrode 35 " or stop electrode 35 " ' (illustrating with white).All electrodes 35 are operatively connected to the electrode selector 34 (only partly illustrate in Figure 4 A in Fig. 4 B and Fig. 4 D exist but not shown) of control unit 23.

Fig. 4 B shows and is provided now ground voltage pulse, one group two electrodes 35 identical with previous pulsed drive electrode 35' ".This causes the liquid part 19' of larger volume back aspirate towards the electrode 35' enabled and cause the liquid part 19' local detachment (referring to double-head arrow) of larger volume to become still to cover two smaller portions 19' of at least two pulsed drive electrode 35' of same electrod-array 21.

Fig. 4 C shows identical with prior pulse formula drive electrode 35' and is provided now ground connection or stops one group of three electrode 35 of voltage pulse (stopping voltage pulse even to facilitate separating effect) ".The liquid part 19' which results in larger volume is now divided into completely, and (the bilateral arrow referring to the left and right) still covers two smaller portions 19' of at least two pulsed drive electrode 35' of identical electrodes array 21.

Being similar to the driving path (as shown in Figure 4) of use three row electrode 35, also can performing the separation (referring to Fig. 5) of smaller size smaller (that is, single drop 19) when using identical particularly preferably droplet manipulation instrument 20.

Fig. 5 shows according to the second preferred embodiment and is similar to the top view that Fig. 3 A has the grid electrode array of the array of rectangular electrode.

In fig. 5, the liquid part 19' of larger volume covers about 6 pulsed drive electrode 35' of identical electrodes array 21.Illustrate with grey at the electrode 35'(enabled), multiple electrode 35 is restricted to earth electrode 35 " or stop electrode 35 " ' (illustrating with white).All electrodes 35 are operatively connected to the electrode selector 34 (only partly illustrate in fig. 5, and to exist in Fig. 5 B and Fig. 5 D but and not shown) of control unit 23.

In order to be separated or the remaining fluid body distributed from larger volume divides the drop 19 of 19', the extra drive electrode 35' enabling identical electrodes array 21 makes liquid part 19' now cover about 7 drive electrode 35'(referring to Fig. 5 B, one-sided arrow to the right).Then, contrary with the direction enabling extra drive electrode 35' in Fig. 5 B, the extra drive electrode 35' enabling identical electrodes array 21 makes liquid part 19' cover now about 7 drive electrode 35'(referring to Fig. 5 B, one-sided arrow to the right).Then, contrary with the direction enabling extra drive electrode 35' in Fig. 5 B, enable another drive electrode 35', and one group two electrodes 35 identical with prior pulse formula drive electrode 35' " be provided ground connection or stop voltage pulse.This causes drop 19 to distribute and the remaining fluid body of larger volume divides 19' to move in different directions (referring to double-head arrow in Fig. 5 C).To one group of two electrode 35 " provide stop pulse even to facilitate desired separating effect.

When utilizing identical particularly preferred droplet manipulation instrument 20 (referring to Fig. 6), the driving path of three row electrodes 35 (such as shown in Fig. 4) is used to be preferably used for distributing less liquid volume with the closest combination arriving the electrode path of the single rows of electrodes (compared with Fig. 3 A) of electrod-array 21, that is, single drop 19.

Fig. 6 shows according to the 3rd preferred embodiment and is similar to the top view that Fig. 3 A has the grid electrode array 21 of the array of rectangular electrode 35 and the electrode path of adjacent electrode array 21.

In fig. 6, about 10 of liquid part 19' coated electrode array 21 of larger volume enable electrode 35'.All electrodes 35 not enabled (such as, be in ground connection or be in stopping potential) of electrode path.Preferably, these 12 electrodes 35 are activated the liquid part 19' collecting this larger volume, and the electrode 35' enabled attracts liquid or at least keeps liquid at stable volume.Most preferably, on the hydrophobic top surface 16 that the liquid part 19' of this larger volume remains on smooth thin polymer film 14 and in gap 17, smooth thin polymer film 14 exposes (compared to Figure 1) to the substrate 22 with at least one electrod-array 21.The electrode of inactive electrod-array 21 will change position and the shape of the liquid part 19' of larger volume significantly.All electrodes 35 are operatively connected to the electrode selector 34 (only partly illustrate in fig. 6, exist in Fig. 6 B and Fig. 6 F but and not shown) of control unit 23.

Be separated to divide 19' from the remaining fluid body of larger volume or distribute drop 19, enabling the first two adjacent driven electrode 35' of electrode path, and forbid previously enabled 12 electrodes 35 (referring to Fig. 6 B) of this array 21.Forbidding represents that 12 electrodes 35 to electrod-array 21 provide ground voltage pulse in the case.This compound action of the first two adjacent driven electrode 35' of electrode path and 12 electrodes 35 of this array 21 causes the liquid part 19' of larger volume to be moved to the left and propagates (the one-sided arrow referring to left) on the first two adjacent driven electrode 35' of electrode path.

Continuously (referring to Fig. 6 C), enable the 3rd drive electrode 35' of electrode path, forbidding the first two adjacent driven electrode 35 ", and also enable one group of 9 electrode 35' of electrod-array.Meanwhile, three electrodes 35 of electrod-array are forbidden ".Forbidding represents the first two adjacent driven electrode 35 to this path in the case " ground connection or stop pulse are provided.This by make drop 19 move to this path the 3rd electrode 35 and make residual liquid part 19' move backward to electrod-array 21 enable electrode 35'(referring to the one-sided arrow in left and right) and cause the first drop 19 to be separated with larger volume liquid part (or distribution).To two adjacent driven electrodes 35 " provide stop pulse even to enhance desired separating effect.Remaining fluid body divides 19' about 9 electrodes of existing coated electrode array.

Then (referring to Fig. 6 D), enable the four-wheel drive electrode 35' of electrode path, forbid the second drive electrode and the 3rd drive electrode 35 ".Forbid the second drive electrode represented to this path and the 3rd drive electrode 35 in the case " ground connection or stop pulse are provided.This causes the first drop 19 to move (the one-sided arrow referring to left) to the 4th electrode 35' in this path.Meanwhile, the first drive electrode 35' of electrode path enables and three of electrod-array electrodes 35 together with adjacent 9 electrode 35' of electrod-array 21 " keep forbidding.This causes the liquid part 19' of larger volume to be moved to the left and propagate (the one-sided arrow referring to left) on the first adjacent drive electrode 35' of electrode path.Three electrodes 35 relative to electrod-array 21 " forbidding force the residual liquid part 19' of larger volume to the electrode 35' near electrode path concentrate.

Hereinafter (referring to Fig. 6 E), enable the 5th drive electrode of electrode path and forbid four-wheel drive electrode.This causes the first drop 19 to move (the one-sided arrow referring to left) to the 5th electrode 35' in this path.Meanwhile, enable first three drive electrode 35' of electrode path, and forbid all electrodes of electrod-array.This causes the liquid part 19' of larger volume to be moved to the left and propagate (the one-sided arrow referring to left) on first three drive electrode 35' of electrode path.

Afterwards (referring to Fig. 6 F), the 5th drive electrode of electrode path and the 3rd drive electrode 35' keep enabling, and four-wheel drive electrode and the first two drive electrode forbidding.This causes the first drop 19 to remain on the 5th electrode 35', is separated (the one-sided arrow referring to left) on the 3rd electrode 35' in this path with the second drop 19.Meanwhile, enable 6 electrodes 35 of this array 21, this causes remaining fluid body to divide 19' to be extracted into electrod-array 21 and sees, therefrom, it now covers about 8 electrodes (the one-sided arrow referring to the right).Before distributing other (the 3rd) drop 19, separated drop 19 at least moves to next drive electrode 35' in each case, and formed as Fig. 6 D the similar situation described.

Especially, about Fig. 4, Fig. 5 and Fig. 6, point out clearly preferably, dielectric layer is arranged on the surface of electrod-array 21.Preferably, dielectric layer is the part of the substrate 22 of droplet manipulation instrument 20 or the part of smooth thin polymer film 14.Deviate from the embodiment shown in Fig. 3 A, gap preferably 17 thereon side is closed by the dielectric layer with hydrophobic surface, drop 19 or liquid part 19' is moved and handles in gap 17 between two hydrophobic surfaces.Gap 17 can partially or even wholly fill not with drop 19 or the miscible fluid of liquid part 19', fluid is preferably selected from air, chemically inactive gas (as N ₂) and non-aqueous liquid (as silicone oil or hexadecane).Substrate 22 can be selected from as materials such as printed circuit board (PCB) (PCB), polymer, glass or combination materials.Preferably, all electrodes 35 are arranged in same level; But, be also susceptible to and electrode 35 is arranged in Different Plane or in different clinoplain.

In the context of this application, from major part 19' " distribution " drop 19, by drop 19 " being divided into " more droplet, and major part 19' " is divided into " some parts and is referred to as and handles drop 19 or liquid part 19', no matter larger liquid part 19' is the need of " suction ".In the context of this application, " associating " (such as each other to make comparatively droplet 19, to match thing for hybrid reaction) or to combine with larger liquid part 19', and " combination " larger liquid part 19' is also referred to as and handles drop 19 or liquid part 19'.In the context of this application, in order to force larger liquid part 19' to control desired direction (such as, to the site of wherein electrode part electrode joint array 21), size that ground reduces motor array 21 is 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 " manipulation array ".The spirit and scope of the present invention comprise belong to claims boundary in and any combination of the rational different embodiments of disclosed electrod-array in the present patent application to those skilled in the art.

Similar Reference numeral refers to similar parts, if they are not discussed in detail especially.

Reference numerals list:

1 biological sample treatment system 26 electrically insulating material

2 container 27 conductive materials

The outside of top side 28 container of 3 containers

The bottom side 29 voltage control of 4 containers

Projection 30 identification device of 5 containers

6, the well shape structure 31 maintenance device of 6' container

Top side 32 system liquid of 7 well shape structures

Opening 33 analysis area of 8 well shape structure bottom sides

9 biological sample 34 electrode selector

10 reaction reagent 35 electrodes

The opening 35' drive electrode of 11 well shape structures

The passage 35 of 12 containers " earth electrode

The aperture 35 of 13 containers " ' stop electrode

14 smooth thin polymer film 36 CPU

Lower surface 37 software of 15 smooth thin polymer films

38 optics in the hydrophobicity of 16 smooth thin polymer films

Surface 39 mesh points

17 gap 40 boxes

18 liquid 41 magnets

19 drop 42 heating element heaters

Liquid part 43 covering of 19' larger volume

20 droplet manipulation instrument 44 power supplys

21 electrod-array 45 side-strut edges

Substrate 46 receiving element of 22 instruments

23 control unit 47 nozzles

24 upper surface of solid substrate d at film and the ends of container comprising biological sample

Distance between 25 setting element sides

Claims

1. a droplet manipulation instrument (20), comprising:

(a) at least one electrod-array (21), it is for by the moistening movement causing drop (19) of electricity;

(b) substrate (22), described at least one electrod-array (21) of its supporting; And

(c) control unit (23), it comprises at least one electrode selector (34) be connected with at least one voltage control (29), and described at least one electrode selector (34) is implemented as to be selected each electrode (35) of at least one electrod-array (21) individually and provides the voltage controlled by described voltage control (29) to described selected electrode (35), described control unit (23) also comprises CPU (36), described CPU (36) for control electrode selector (34) and described voltage control (29) to select at least one electrode (35) individually and to provide individual voltage pulse at least one selected electrode (35), described individual voltage pulse is selected from and comprises driving voltage, the cohort of ground voltage and stopping voltage, thus described selected electrode (35) is defined as drive electrode (35'), earth electrode (35 ") or stopping electrode (35 " '), wherein

D () described control unit (23) can by limiting in the drop (19) of the more than one electrode (35') of a covering electrod-array (21) and the liquid part (19') of larger volume the mobile route guided of at least one by two or more drive electrode (35') groups subsequently substantially selecting described electrod-array (21) simultaneously, and in these selected drive electrodes (35'), each provides drive voltage pulses along described path; And

E () control unit (23) is implemented as to organize to two or more electrodes (35) adjacent or identical with pulsed drive electrode (35') and substantially provides ground connection simultaneously or stop voltage pulse.

2. droplet manipulation instrument (20) according to claim 1, is characterized in that, the described electrode (35) of described electrod-array (21) is arranged as orthogonal grid.

3. droplet manipulation instrument (20) according to claim 1, is characterized in that, the described electrode (35) of described electrod-array (21) shows as square or round-shaped substantially.

4. droplet manipulation instrument (20) according to claim 1, is characterized in that, the described electrode (35) of described electrod-array (21) shows as hexagonal or leg-of-mutton shape substantially.

5. droplet manipulation instrument (20) according to claim 1, it is characterized in that, the described electrode (35) of described at least one electrod-array (21) is positioned to the described upper surface of described electrode (35) and the described upper surface flush alignment substantially of described substrate (22) relative to described substrate (22), and/or the described electrode (35) of described electrod-array to be positioned in described substrate (22) and to be closed by described substrate (22).

6. droplet manipulation instrument (20) according to claim 1, it is characterized in that, its be implemented as hold for larger volume process at least one container (2) and there is at least one smooth thin polymer film (14) of hydrophobic top surface (16).

7. droplet manipulation instrument (20) according to claim 6, it is characterized in that, it comprises at least one heating element heater (42), the humidity province of the temperature that described heating element heater (42) provides at least one to have to limit in advance on the described hydrophobic top surface described smooth thin polymer film (14).

8. droplet manipulation instrument (20) according to claim 1, it is characterized in that, it is implemented as at least one box (40) that accommodation comprises container (2) and the smooth thin polymer film (14) with hydrophobic top surface (16).

9. droplet manipulation instrument (20) according to claim 8, it is characterized in that, it comprises at least one heating element heater (42), the humidity province of temperature of described heating element heater (42) for providing at least one to have on the hydrophobic top surface (16) of described smooth thin polymer film (14) to limit in advance

10. a biological sample treatment system (1), comprising: droplet manipulation instrument (20) according to claim 1, and described biological sample treatment system (1) also comprises:

A () is for the container (2) of larger volume process, it has top side (3) and bottom side (4) and comprises at least one well shape structure (6), described well shape structure (6) is opened wide in described top side (3) to locate biological sample (9) and/or reaction reagent (10) wherein, at least one well shape structure (6) wherein said comprises bottom side (8), described bottom side (8) has at least one opening (11), described container (2) also comprises passage (12), described passage (12) connects the aperture (13) on the described opening of described well shape structure (11) and the described bottom side of described container (2), and the described bottom side (4) of wherein said container (2) comprises distribution projection thereon (5), and

(b) smooth thin polymer film (14), it has lower surface (15) and hydrophobic top surface (16), described hydrophobic top surface (16) keeps described bottom side (4) d with a certain distance from described container (2) by described projection (5), when described container (2) to be positioned on described smooth thin polymer film (14) with its projection (5) against described smooth thin polymer film (14), described distance d limits at least one gap (17)

Wherein said container (2) and described smooth thin polymer film (14) are reversibly attached on described droplet manipulation instrument (20).

11. biological sample treatment systems (1) according to claim 10, is characterized in that, described smooth thin polymer film (14) is embodied as the smooth thin polymer film of electric insulation.

12. biological sample treatment systems (1) according to claim 10,

It is characterized in that, when described container (2) and described smooth thin polymer film (14) are reversibly attached on described droplet manipulation instrument (20), the described lower surface (15) of described smooth thin polymer film (14) is against described at least one electrod-array (21), described system (1) allows at least one drop (19) to be displaced to by the described passage (12) of described container (2) the described hydrophobic top surface (16) of described smooth thin polymer film (14) and in described at least one electrod-array (21) top from least one well shape structure (6) described,

And wherein, described droplet manipulation instrument (20) is implemented as and controls the movement that guided of described drop (19) on the described hydrophobic top surface (16) of described smooth thin polymer film (14) by electricity is moistening and processes described biological sample (9) there.

13. biological sample treatment system (1) according to claim 12 or boxes (40), is characterized in that, described smooth thin polymer film (14) is embodied as the smooth thin polymer film of electric insulation.

14. 1 kinds of methods utilizing the biological sample treatment system (1) described in claim 10 to process biological sample (9),

Wherein, described container (2) and described smooth thin polymer film (4) are reversibly attached on described droplet manipulation instrument (20), make the described lower surface (15) of described smooth thin polymer film (14) against at least one electrod-array described, and at least one drop (19) to be displaced to by the described passage (13) of described container (2) the described hydrophobic top surface (16) of described smooth thin polymer film (14) and in described at least one electrod-array (21) top from least one well shape structure (6) described, and

The electricity wherein controlled by described droplet manipulation systems (20) is moistening and the movement guided on the described hydrophobic top surface (16) of described smooth thin polymer film (14) processes described drop (19).

15. methods according to claim 14, it is characterized in that, described container (2) and described smooth thin polymer film (14) are set to box (40), described box (40) comprises described container (2) and described smooth thin polymer film (14), and described container (2) and described smooth thin polymer film (14) are attached to each other by gluing together or weld.

16. methods according to claim 14, is characterized in that, described container (2) and described smooth thin polymer film (14) are set to remain to the separate part of just assembling when being about to start to process biological sample (9).

17. methods according to claim 14, is characterized in that, in order to precipitation of liquid droplets (19) and the liquid part (19') of described larger volume, and described control unit (23):

I () is to initial drive electrode (35') and provide drive voltage pulses along described electrode path at the electrode (35') at described initial drive electrode (35') rear and therefore make described drop (19) move in a first direction; And

(ii) described two or more drive electrodes (35') group to described electrod-array (21) provides drive voltage pulses to make the liquid part (19') of described larger volume move up in second party opposite to the first direction.

18. 1 kinds of methods utilizing the biological sample treatment system (1) described in claim 12 to process biological sample,

Wherein, the electricity controlled by described droplet manipulation systems (20) is moistening and the movement guided on the described hydrophobic top surface (16) of described smooth thin polymer film (14) processes described drop (19).

19. methods according to claim 18, it is characterized in that, described container (2) and described smooth thin polymer film (14) are set to box (40), described box (40) comprises described container (2) and described smooth thin polymer film (14), and described container (2) and described smooth thin polymer film (14) are attached to each other by gluing together or weld.

20. methods according to claim 18, it is characterized in that, described container (2) and described smooth thin polymer film (14) are configured to separate part, and described separate part will remain when will start to process biological sample (9) assembled.

21. methods according to claim 18, is characterized in that, in order to precipitation of liquid droplets (19) and the liquid part (19') of described larger volume, and described control unit (23):