CN1950506A - Nucleic acid purification chip - Google Patents
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- CN1950506A CN1950506A CN 200480039520 CN200480039520A CN1950506A CN 1950506 A CN1950506 A CN 1950506A CN 200480039520 CN200480039520 CN 200480039520 CN 200480039520 A CN200480039520 A CN 200480039520A CN 1950506 A CN1950506 A CN 1950506A
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Abstract
The present invention provides a novel system of extracting and purifying nucleic acids (DNA, RNA, etc.) from cellular material like blood. Such a system of extraction and purification relies on novel monolithic microfluidic devices and methods of using these devices. Such devices comprise numerous components, monolithically-incorporated on an single chip, and further comprising novel nucleic acid binding materials. The present invention is also directed to method of preparing such novel nucleic binding materials.
Description
The cross reference of related application
The right of priority that the application requires to enjoy in is that on December 30th, 2003 submitted to, sequence number is 60/533,297 following U.S. Provisional Patent Application.
Technical field
Relate generally to biotechnology of the present invention relates to micro-fluidic (microfluidic) method and apparatus based on chip that is used to extract with purification of nucleic acid particularly.
Background technology
The genomics field that is widely used is as crime analysis, clinical diagnosis etc.It is applied to the field such as agricultural, health care, environmental monitoring and study of pharmacy etc.In most of the cases, genomic dna obtains from the white corpuscle from human blood.The method that is used to obtain this DNA requires usually from biogenetic derivation isolating nucleic acid separately.For purifying people DNA from blood, when beginning, DNA is limited in the white corpuscle.In order to extract and this DNA of purifying, must open (cracking) cytolemma with one or more different methods, these methods comprise chemistry, osmotic pressure, heat, electricity and physical method.
The current techniques that obtains this DNA in hospital or laboratory from blood is unusual effort and to require a lot often be manual step still.Step in the past is the cracking hemocyte, and wherein cell is opened by breaking and its nucleic acid is released in the solution and respective regions that can be used for purifying.The method of membranolysis and release nuclear content means that other biomolecules also will exist in solution.In these molecules some combine with nucleic acid in the mode of not expecting as protein and metal composite (for example oxyphorase), and then disturb typical subsequent processing steps such as pcr amplification (polymerase chain reaction).Therefore, need be from chip material the step of DNA isolation.Afterwards, the requirement of any dna purification system all is must isolating nucleic acid, these inhibitor of flush away simultaneously.All be manual operations as all these step 1, need the plenty of time.Therefore, exploitation can be carried out these operations automatically, use still less that the certain methods of sample is important.
Production can will be useful with the genomic dna that purifying is provided as the micro-fluidic chip of exporting with people's whole blood of automated manner processing and processing microlitre μ l (or littler) volume.It would be desirable that this DNA specimen preparation micro-fluidic chip is the device of integrated micro mixer, micro-filter, microreactor etc.Output should have and similar productive rate and the purity of using now of conventional macrosystem.The current trend of Protocols in Molecular Biology requires testing method to keep good yield results but reduces test duration, reagent volume and cost.And the automatization level increase is more common.Also have the requirement of miniaturization, make test need not be limited to centralab and can be portable, for use in rig-site utilization or point-of care test.
For chip design, repeatedly disclose one strainer, valve, mixing tank and reactor assemblies in the past in 10 years, but special application has been arranged for these designs of different assemblies, repel them and be used for the combination of general purpose.That realizes all these assemblies that the DNA sample preparation/purification requires integratedly is still a kind of big challenge.Although be disclosed before integrated a plurality of assemblies on the polymer substrate, these generally are used for microarray, PCR system.The unique known embodiment of complete micro-fluidic scheme technology that is used for carrying out from blood the DNA sample preparation/purification is open by people such as Kim.See Kim et al., " A Disposable DNA Sample Preparation Mierofluidie Chip forNucleic Acid Probe Assay, " IEEE-MEMS 2002, pp.133-136.It is integrated in (substrate comprises mixing tank) on the PDMS substrate with silicon strainer and glass reactor.The scheme of people such as Kim report is the mapping mode of integrated micro-mechanical assembly on polymer substrate.Different assemblies is binding substances (binder) (by the glass manufacturing) and strainer (being made by silicon, nickel and PDMS).Connect substrate and be different from conventional interconnect (interconnect) substrate, because interconnection forms micro mixer.
The current operational aspects typical that is used for nucleic acid purification relates to a lot of steps, and wherein plurality of reagents is added in the sample, and sample by centrifugal with precipitation separation component and solution.This method is very complicated, is still manual carrying out under a lot of situations.Because some surfaces of the known selective binding of nucleic acid, big quantity research concentrates on the interaction of these types.Have been found that to be applicable to nucleic acid bonded kinds of surface, and this combination technology uses silica-gel bead combination, coupling antibody, silane, nucleic acid, polylysine, poly--T-DNA, some bronsted lowry acids and bases bronsted lowry.
Several method and device have been developed recently, to attempt to improve the genomic analysis system in the micro device.U.S. Patent No. 6,379,929 disclose the method and composition of isothermal amplification of nucleic acid in little process substrate.The method and composition of analyzing isothermal duplication nucleic acid in little process substrate is also disclosed.Little process substrate that provides and isothermal duplication and detection method intention is used for multiple diagnostic method, especially be characterized as those of disease-related that gene order or genetic expression changes.Yet these only pay close attention to nucleic acid amplification, and do not consider DNA extraction and purifying.
U.S. Patent No. 6,368,871 have described the apparatus and method that are used for operating fluid sample material (as particle, cell, macromole, as protein, nucleic acid or other composition).Described device comprises the substrate with a lot of microstructures (pillars) and this structure upper nonconductive Film.Apply voltage to described structure and induce material in the sample separation.Described apparatus and method can be used for using widely, as dielectrophoresis (DEP) or make target material and fluid sample in other material separation.These technology are used column construction, apply voltage to it and are beneficial to mix.
U.S. Patent No. 6,168,948 provide integrated diagnostic nucleic acid device of miniaturization and system, and it comprises the nucleic acid extraction district, and this district comprises the nucleic acid binding site.The miniaturization nucleic acid extraction of this patent disclosure and sample refining plant comprise porous percolation deformable plug and are used for bind nucleic acid, or have the structure of sample binding site in cell.Form later on or add this stopper forming microfluidic channel, make it to become assemblage or in-situ synthetic method.Reason for this reason, it can not be described to is whole, or is described as having the batch that is produced by global design and makes advantage.
Summary of the invention
The present invention relates to be used for extract and the apparatus and method of purify DNA from cell.This apparatus and method provide from cell material and systematically take out and isolating nucleic acid.Typically, cell material obtains from blood (being white corpuscle).
In some embodiments, the present invention relates to micro fluidic device.Device of the present invention the design and textural be all-in-one-piece.In some embodiments, described micro fluidic device comprises inlet, mixing tank, reaction chamber, the nucleic acid bond material of silicon substrate, introducing microliter amount and Geng Duo material and the outlet of taking out material from device.
The bond material selectivity combines with nucleic acid.In some embodiments,, use plasma etch process plasma etching silicon oxide surface (Cement Composite Treated by Plasma) then by at first using thermal oxide art breading silicon substrate, thus the preparation bond material.In other or other embodiment, make bond material of the present invention by on substrate, depositing based on the silicon oxide of silane with plasma enhanced chemical vapor deposition (PECVD) technology.
In some embodiments, the present invention relates to the monoblock type micro fluidic device made with novel process.This device provides from cell material and to extract and purify DNA, but with new, cost efficient manner structure, and comprises with the new and new bond material produced of mode efficiently.
In some embodiments, the present invention relates to handle the method for nucleic acid.In these embodiments, make cell material break (cracking) with the release content, and the nucleic acid moiety in the separate content thing.These methods typically use chemical technology with the lysing cell material.Part is separated in conjunction with the realization nucleic acid content with bond material by selectivity under controlled condition, and wherein said bond material is a new bond material of the present invention.
The present invention is different from those of people such as Kim, and its design is all-in-one-piece and on silicon.This means the assembling that does not have multiple assembly.It also means by the most of fluid stream in the microchannel being maintained in the substrate plane (low dead volume), only makes that entrance and exit stream is transited into and the vertical stream of substrate plane (high dead volume), thereby makes dead volume littler.Integral body is integrated into also provides inter-module heat-insulating possibility on the silicon, cause reducing steady state power consumption under higher temperature, uniform temperature spectrum in the reactor is by changing the heat sink quick change system temperature that is connected with substrate, rapid thermal cycles is with the system that is made up of different simultaneous temperature districts.
Feature of the present invention has roughly been summarized in the front, thereby following detailed description of the present invention can better be understood.Additional features of the present invention and advantage will be described below, and they form the theme of claim of the present invention.
Description of drawings
In order to understand the present invention and advantage thereof more fully, with reference to following explanation also in conjunction with the accompanying drawings, wherein:
Figure 1A and B are the system schematic that illustrates two embodiments of the present invention, and wherein difference between the two is there be (B) or lack (A) of filter assemblies;
Fig. 2 is the schema according to the universalization method of extraction of the present invention and purification of nucleic acid;
Fig. 3 description taken in conjunction and wash-out mechanism, its amplifying nucleic acid under high salt condition (A) combine with bond material, and under low-salt conditions (B) wash-out;
The plan cross-sectional view of Fig. 4 major electrical components one embodiment of this invention, its design has silicon-glass integrated structure;
Fig. 5 illustrates the chip of some embodiments of the present invention, comprises a lot of assemblies;
Fig. 6 A and B explanation gel electrophoresis plate, the wherein binding ability of one of bond material that has four kinds of different preparations of (or lacking) reflection of band in given swimming lane;
Fig. 7 illustrates the relation between the temperature and joint efficiency according to embodiments of the present invention; With
Fig. 8 explanation comprises the nucleic acid elution efficiency of device under differing temps by the bond material of different methods and/or Processing of Preparation.
Embodiment
The present invention relates to from the apparatus and method of cell extraction and purify DNA (or other nucleic acid).In general or separate, these apparatus and method can form to provide and extract and the system of these nucleic acid of purifying.
Provide to give a definition with the better the present invention of understanding.
" nucleosides " be with ribose or ribodesose with glycosidic link bonded purine or pyrimidine." Nucleotide " is the phosphoric acid ester of nucleosides.Oligonucleotide through phosphodiester bond connect, up to the linearity " sequence " of 20 Nucleotide or " monomer "." nucleic acid " is through 3 ', 5 ' phosphodiester bond linear polymer that connect, Nucleotide (as oligomer, but longer).In the thymus nucleic acid " DNA ", glycosyl is a ribodesose, and nucleotide base is VITAMIN B4 (A), guanine (G), thymus pyrimidine (T) and cytosine(Cyt) (C).Yeast Nucleic Acid " RNA " has as the ribose of glycosyl and identical nucleotide base, and just uridylic replaces thymus pyrimidine.Single stranded DNA have base A, G, T and C's " sequence ".For example when forming duplex, keep this secondary structure together by the hydrogen bond between the base on the adjacent chain.Please note in this base pairing the total and T bonding of A, the total and G bonding of C.
The DNA that " genomic dna " found in organism " genome " (being genetic stockss all in the karyomit(e) of particular organisms); Its size generally is expressed as the base pair sum, and passes to the offspring as the essential information of existence.This speech is used to distinguish other type DNA, as the DNA that finds in the plasmid." genomics " expression genome research, it comprises genomic mapping, gene sequencing and gene function.
The relative order of base pair in dna fragmentation, gene, karyomit(e) or the full genome is determined in " gene sequencing " expression.The key of a lot of gene order surveying methods is electrophoretic separation techniques, as " gel electrophoresis ", " capillary electrophoresis " and " disc electrophoresis ".
" PCR ", it is the polymerase chain reaction, it is the system of amplification in vitro DNA, wherein rise in the presence of acid and the Taq polysaccharase (heat-stable DNA polymerase), will be added in the target dna with two synthetic oligonucleotide primer things (a corresponding chain of primer) of two regional complementarities of target dna to be amplified at excessive deoxidation nuclear.In series of temperature circulation, DNA repeats to be denatured, annealed on the primer and from the primer extension subchain.Because subchain is used as template in follow-up circulation, amplification takes place with exponential manner.
" active-ion-etch " (RIE) or " dark active-ion-etch " (DRIE) is represented so a kind of technology, wherein radio frequency (RF) or microwave radiation coupling are gone into low-pressure gas and gas molecule are dissociated into have more active material, treat that etch substrate (typically based on silicon) is biased to induce ion bombardment.Usually use the compound of carbon containing (C) and halogen such as fluorine (F), chlorine (Cl) or bromine (Br) as gas.When compound dissociates in plasma body, generate high reactivity halogen atom or halogen compounds and can deposit on the substrate and block highly active polymkeric substance.Ion is accelerated to by applying or induce biasing on the etched substrate, thereby removes the polymkeric substance perpendicular to the substrate surface of ion motion direction, and the substrate surface that polymer coating is parallel with the ion motion direction is also blocked these surperficial etchings.Ion bombardment can also activate or quicken the chemical milling reaction.Therefore RIE has with higher relative rate etching with the vertical surface of ion motion direction, with the ability on the surface parallel with the ion motion direction than the low relative speed etching, causes anisotropic etching.
" MEMS (micro electro mechanical system) " (MEMS) comes from micro mechanical structure (containing moving portion) and microelectronic integrated.
According to the present invention, " thermal oxide technology " relates at oxygen (O
2) and/or steam (H
2O) exist down with silicon | wafer | be heated to 600 ℃-1250 ℃ temperature.The temperature of this rising strengthens the oxygenant diffusion, and causes oxide compound significantly to be thicker than the 2nm native oxide that silicon oxidation produces in the air at room temperature.
" chemical vapour deposition " (CVD) represented to deposit from the material of gas chemistry precursor.
According to the present invention, " monoblock type " or " whole integrated " meaning be meant all component with the current techique design, be manufactured on the common substrate simultaneously and guide fluid stream to be positioned at the plane on substrate wafer surface.
With reference to figure 1, can in system schematic, observe the present invention.Shown in Figure 1A, multiple input thing (blood, cracking agent, high level salt solution, alcohol, air and low salts solution) is introduced micro fluidic device of the present invention by valve.At first blood and cracking agent are introduced mixing tank or mixing section by valve.Through mixing, the break cytolemma of white corpuscle (WBC) and red corpuscle (RBC) in the blood of cracking agent, the nucleic acid material that release WBC contains.After the cracking, valve leads nucleic acid (with other waste material of blood and hemocyte) in conjunction with the chamber under high salt condition, wherein comprises the binding substances of being made up of bond material.High level salt solution stream guarantees that the nucleic acid selectivity combines with bond material, but waste material passes through and flows out as refuse.Nucleic acid still with bond material bonded situation under, with alcohol (as ethanol) rinsing binding substances, dry air (forced convection) is randomly used the less salt eluant solution at last then.Therefore, blood contacts with cracking agent, WBC and RBC cracking in mixing tank together.Entocyte discharges, and DNA (from WBC) combines with binding substances.In this embodiment, in conjunction with before cell can be only with a mixing tank cracking, thereby the program of realization cracking, combination and wash-out.
Scheme as an alternative, described system can be used the strainer that can catch white corpuscle but allow red corpuscle and other material to pass through before introducing cracking agent.This embodiment is shown in Figure 1B.Wherein blood is introduced into and is mixed together with phosphate buffered saline buffer (PBS), passes through strainer subsequently.Rinsing is removed after RBC and other material, as above-mentioned cracking WBC and import in conjunction with the chamber to be further purified.Therefore this embodiment allows to carry out preliminary purification before being exposed to bond material.
Observe by different way, the present invention extracts from cell material and the method for purification of nucleic acid.This method generally comprises series of steps.With reference to figure 2, step 2001 is hemodilution steps, with reduce viscosity and make the microchannel of the easier described device below of mixture and microchamber in flow.Can realize dilution by adding phosphate buffered saline buffer (PBS) or other similar solution.Step 2002 is cleavage step, and wherein cracking agent is added in the dilution blood solution and discharges the nucleic acid material with the ruptured cell film and from WBC.Please note that DNA only is found in the WBC in the blood, because RBC is seedless.These cracking agents chemical cracking agent typically (chemical cracking) also can be used but other type cracking, as ultrasonic degradation, thermo-cracking, electric cracking and Mechanical Crushing cytolemma (being called mechanical lysis).Step 2003 is the steps that under the high content of salt condition nucleic acid that discharges are attached on the bond material.This realizes by the salts contg (being concentration) in the careful control solution with by the mating surface (bond material) that special processing is provided.The nucleic acid that discharges is directed to bond material/chamber through the combination of fluid stream and valve.Step 2004 is elution step, when the salts contg condition is changed to the condition of lower salt content or water and realize.
Fig. 3 explanation is attached to bond material or substrate under high salt condition, the process of wash-out (relating to cohesive process) under low-salt conditions.Also can increase other step, as filtration, washing, rinsing and drying.These washing steps can comprise the washing of high salt so that in conjunction with stronger, alcohol washing removing fragment or other waste material, and randomly dry air to remove alcohol.These methods are used the new device of representing embodiment of the present invention with its oneself right.
In some embodiments, the present invention relates to micro fluidic device.In some embodiments, described micro fluidic device comprises silicon substrate, is used for introducing the inlet, mixing tank, reaction chamber, nucleic acid bond material of microliter amount material and from being used for the outlet that device takes out material.These devices typically comprise by the integrated assembly of integral body.
Described bond material selective binding nucleic acid.In some embodiments, by at first using thermal oxide art breading silicon substrate, prepare bond material with plasma etch process plasma etching silicon oxide surface then.In other or other embodiment, make bond material of the present invention on the substrate by depositing to based on the silicon oxide of silane with plasma enhanced chemical vapor deposition (PECVD) technology, use or do not use follow-up plasma etch process.In other or other embodiment, the chemical vapor deposition (CVD) technology of other type such as positive silicic acid tetraethyl ester (TEOS) can be used for cvd silicon oxide, use or do not use follow-up plasma etch process.
In some embodiments, the present invention relates to introduce the micro fluidic device of new functional design.These devices provide from cell material and to extract and purify DNA, but with new, cost efficient manner structure.These devices comprise integral design and new bond material.
Totally observe as the miniflow sample disposal system, apparatus and method of the present invention provide the monoblock type chip that is designed for nucleic acids for preparation (as purifying).More specifically, the invention provides from blood (generally be human blood, but also can be the blood of other Mammals and nonmammalian) extraction and purify DNA and/or RNA (nucleic acid).For device of the present invention, functional microfluidic components typically is integrated on the single substrate.The function that these assemblies have comprises mixing, filtration, combination etc.Successfully be used for DNA extraction although the present invention has proved, its application is not limited to DNA extraction, can be used for can selectivity directly or close the purifying of any material of binding substances of the present invention by centre mediation material or intermolecular access node.
As used herein, whole integrated such device that provides, wherein all component is by with the common technique design and use fluid stream in the wafer surface plane, except fluid intake and exit stream can be perpendicular to wafer surface or in this surface.This integral design of assembly provides easier manufacturing and operation.
The present invention with respect to the advantage of currently used macrosystem is: unattended operation, small size and watt consumption are convenient to portable application.Advantage with respect to non-integral microcosmic scheme is: assemble easily and encapsulate; Littler dead volume, whole size; Improved thermal design (because the thermal properties of silicon and how much micromechanics possibilities); Add other sensing function alternatively, be connected because all component can both easily be crossed the interface with the electrical readout link tester; Make components of system as directed or full-automation alternatively.
Surface condition, surface-area, the chemicals, pH and the temperature that flow spectrum, use---all the utmost point influence the ability of extraction of the present invention and purification of nucleic acid.Therefore, the inventive method provides the careful control to these parameters.This design and operation for apparatus of the present invention is actual especially.
The invention still further relates to provide from cell material and extract and the monolithic microfluidic devices of purification of nucleic acid.Fig. 4 shows the plane view cross-sectional schematic diagram of apparatus of the present invention embodiment, and wherein design has silicon-glass integrated structure.In this specific embodiment, device 400 forms in substrate 405, and is covered by chip glass 401.Coverture 401 is transparent, allows light to reach passage 406.402 and 403 provide the silicon dorsal part opening of entrance and exit.By make such as stop up, flow velocity, flow velocity homogeneity, fluid interface and the fluid interface item by the process of system of striding passage width can detect, the light in-out apparatus makes it possible to the optical sensing apparatus performance.Light can in-out apparatus also make it possible to through fluorescence, photoabsorption maybe can be by other type optical technology of glass window the optical detection reactions product.
According to the present invention, for the typical devices embodiment, reader relates to device 500 shown in Figure 5.According to Fig. 5, blood and phosphate buffered saline buffer (PBS) can be respectively through the 501 and 503 introducing mixing tanks 502 that enter the mouth.Fluid flows directly into strainer 504 then, catches WBC at this, but allow RBC by and flow out through exporting 508.Please note that strainer 504 can omit, as mentioned in the above system description (seeing Figure 1A).After this, lysis buffer is filtered the WBC that device is caught through enter the mouth 505 introducings and cracking.After the WBC cracking, the DNA of release is by strainer 504.At this moment, valve 506 will be operated closes passage 507.DNA and other composition and solution will enter binding substances 513 and with the binding substances surface bonding.Nucleic acid is with after binding substances 513 combines, and can 509 pump into high level salt solution and makes in conjunction with stronger by entering the mouth, and 510 pumps into alcohol with the washing binding substances and make the binding substances cleaning through entering the mouth then, and this helps the nucleic acid in the binding substances.After this, optional can use the dry binding substances of forced convection, and especially dry (promptly removing) alcohol is because alcohol directs the influence to the nucleic acid quality of nucleic acid purification afterreaction such as PCR.Last step is with released dna through the low salts solution of 512 pumps that enter the mouth.At this moment, valve 515 will be operated closes passage 516.Then, DNA will be from exporting 514 by wash-out.Randomly, can increase heat insulation groove 519 and resistance heater 520 with heat affecting cracking/combination/elution process.In addition, can or force pressurized air 511 to produce above-mentioned forced convection through vacuum with dry binding substances by entering the mouth.Compare the faster and easier control of this method with traditional natural convection.
Processing sequence and sample (blood) and/or reagent are introduced the position and are had handiness.In some embodiments, sample and reagent can introduce the point of crossing, microchannel, directly enter reactor or by or a plurality of mixing tanks---this depends on mixed-level that needs and the flow velocity that relates to.
In some embodiments, use the traditional MEMS manufacturing technology at silicon wafer or the silicon/glass wafer set manufacturing chip that closes.All component such as mixing tank, strainer and binding substances are positioned on the chip, with some function that realizes that specimen preparation, processing and purifying need.These assemblies comprise passage, input terminus, output terminal, reactor, mixing tank, strainer, binding substances, RTD and resistance heater.And the isolation features on the substrate provides independently component heat operation.
The importance of said apparatus assembly is the binding substances design.Binding substances is an assembly of directly being responsible for DNA or RNA separation and purifying.When comprising the solution process binding substances of nucleic acid and other fragment or refuse, binding substances is with selective binding nucleic acid and other material is flow through.In step subsequently, binding substances will discharge nucleic acid under certain through engineering approaches condition.Therefore, the binding substances surface is important in this chip.For this surface, the present invention can use one or more several different designs, includes but not limited to silica-gel bead combination, acid, alkali, silane, polylysine, coupling antibody, nucleic acid and poly--T DNA.In some exemplary, according to manufacturing process, the preparation of binding substances chamber surface can comprise with thermal oxide technology uses CHF subsequently
3And O
2Plasma etch process, or as an alternative scheme with plasma enhanced chemical vapor deposition (PECVD) based on the silicon oxide of silane.These technologies produce nucleic acid combination and wash-out aspect surface of good.Use this surface, do not need extra treatment step that chip surface is carried out modification.Plasma treatment step can realize in the wafer front side nitride stripping technology, and this is which kind of mode chip manufacturing necessary step no matter.
In some embodiments, binding substances (bond material) design has the surface of Cement Composite Treated by Plasma, considers also how temperature influences cohesive process.In some embodiments, it has the temperature sensor of well heater and binding substances outside, so that difference and/or uniform temperature to be provided, produces better in conjunction with condition.Also considered thermal isolation.In the case, it can be partial making the heat that applies by isolation features on the substrate, thereby makes the substrate different piece be in differing temps.
The integrated all samples preparation process of miniflow sample disposal system of the present invention is as mixing, filtration, combination, wash-out and independent thermal control.It is to have proved the general-purpose system that successfully is used for DNA extraction, but its application is not limited to DNA extraction.The whole integrated all component that means has been positioned at the fluid stream on wafer surface plane with common technique design and use.Introduced isolation features, make the chip different zones be heat independently.
The surface of bond material is the important factor of above-mentioned joint efficiency.Another important factor of bonded is a surface-area.Can use different methods to increase combined surface area.A kind of such method is to introduce some micro-machined features, and as increasing the number of pillars (microstructure), this increases the vertical area of binding substances surface-area.In order to confirm, design of these pillars (or other similar microstructure) and arrangement (placement) must be considered easier mobile pattern, still less bubble, lower obstruction level and other associated problem.Another kind method is to make the surface more coarse to increase surface-area by chemistry or physical method.Surface roughening can be used to increase and can be used for the bonded surface-area on chip glass 401 or the silicon wafer 405.The technology of roughening glass surface comprises active-ion-etch, plasma etching and wet etching.Under all these situations, can increase surfaceness, but uneven projection may occur, as during etching by natural little those that glass surface produces of covering of bubble, reaction product or involatile constituent in the glass.Similar technology can be used to increase the surfaceness of silicon.In addition, by regulating technology to increase the natural bathtub construction (scalloping) that produces in the bosch DRIE technology, degree of depth active-ion-etch (DRIE) can be realized the silicon face roughening at channel side wall later.
The present invention comprises with respect to other advantages of the commercial extraction test kit that extracts DNA from cell of current use: (each sample～2ml requires (each～1 μ l of extraction is than each～3001 μ l of extraction) and extraction time to reduce (each＜2 hours ratios at every turn～1 day) than each sample～400ml), littler blood sample to reduce the reagent requirement.
Provide following examples so that specific embodiments of the present invention to be described.It will be understood by those skilled in the art that disclosed method is only represented exemplary of the present invention in following examples.Yet according to present disclosure, it will be recognized by those skilled in the art that under the situation that does not depart from the spirit and scope of the invention, can much change and still obtain the same or analog result described specific embodiments.
Embodiment 1
Present embodiment illustrates that miniflow nucleic acid purification chip of the present invention can be manufactured.This chip can be by the following steps manufacturing:
Step 1: naked silicon chip obtains the oxide compound of the about 0.5 μ m of thickness through the thermooxidizing oxidation.The stoichiometric silicon nitride layer of then that 0.15 μ m is thick low-pressure chemical vapor deposition deposits on the silicon oxide.
Step 2: the wafer of covering abovementioned steps then is used for DRIE.Mask layer can be a photoresist material, but the RIE degree of depth this may need to become another kind of material when surpassing about 40 μ m.After being etched with, removes silicon photoresist material.
Step 3: with the passage of DRIE at the front side etch abovementioned steps wafer of silicon wafer.
Step 4: next, selectivity is covered the dorsal part of abovementioned steps silicon wafer with photoresist, with the opening of active-ion-etch etching dorsal part fluid intake and outlet in silicon nitride and silicon oxide.Remove photoresist material then.
Step 5: then, the silicon wafer front side is protected in a side chuck, and in potassium hydroxide solution, passed through the DRIE etched wafer, arrive the bottom of the etched feature in front side until the dorsal part hole.
Step 6: then through plasma etching (CHF
3And O
2) remove the silicon nitride of wafer front side, the silicon oxide below exposing.
Step 7: then, the thick Al layer of about 1 μ m is splashed on the chip glass.
Step 8: selectivity is covered the Al of sputter with photoresist then, and carries out etching with the aluminium Wet-etching agent based on phosphoric acid of standard, and is used as semi-conductor industry.
Step 9: remove photoresist material from chip glass at last, the chip glass anode is attached to the front side of silicon wafer.In conjunction with before chip glass is aimed at silicon wafer.
Gained two plates device through above-mentioned technology manufacturing comprises the microfluidic channel that is etched in the silicon wafer front side.These hole and exterior through being etched in the silicon wafer dorsal part.The fluid channel size changes to wide above 5mm from 2 μ m are wide.The typical about 10 μ m of passage are wide.Typical strainer is formed the wide and dark about 10 μ m of pillars by about 2-3 μ m pillars pillars at interval.Add cap wafer closed channel with glass.
In other embodiments, connect microfluidic structures and can produce by on chip glass, holing before combining at anode with the hole of outside.Do not need above-mentioned steps 5 in this case, wherein the silicon wafer front side is protected in a side chuck, and passes through the DRIE etched wafer in potassium hydroxide solution, until the bottom of dorsal part hole arrival in the etched feature in front side.In replacement scheme or other embodiment, do not need aluminum layer steps (step 7 and 8), because aluminium lamination need not be used for all embodiments of the present invention as well heater, temperature sensor or flow-sensint unit.In some or other embodiment, by opening the big zone of silicon dorsal part, close the instrument pad of coming in and going out even as big as making toe-in, thereby realize being connected with the pad of aluminium lamination.
At last, it should be noted that reaction chamber and the typical case of filtration chamber have about 0.4 μ l volume, the mixing apparatus 0.15 μ l dead volume of having an appointment.Dorsal part hole typical case is the 1mm * 1mm opening of wafer backside, has and anisotropic wet etch＜100〉54 ° of slopes of characteristic that silicon is relevant.
Present embodiment is used for illustrating the influence of the bond material of the thermal oxide manufactured that further Cement Composite Treated by Plasma is used embodiment of the present invention.
In this embodiment, estimate the purification efficiency of the bond material of thermal oxide method generation by the eluate that compares four kinds of different bond materials:
-independent thermal oxide.
(" Piranha " comprises 3: 1 the vitriol oil to-thermal oxide+hydrogen peroxide/sulfuric acid: 30% H
2O
2) clean.
-thermal oxide+plasma etching.
-thermal oxide+plasma etching+hydrogen peroxide/sulfuric acid (" Piranha ") cleans.
Wherein Cement Composite Treated by Plasma comprises CHF
3+ O
2Environment.
Under each identical test condition, from liquid condition such as 6M guanidine hydrochloride solution DNA is combined with various bond materials, then rinsing material in the 6M of cleaning guanidine hydrochloride solution with typical high salt for four kinds of different binding substancess that prepare.Under low-salt conditions, use 1 * TE damping fluid (10mM Tris-Cl and 1mM EDTA) eluted dna then and and use pcr amplification.
Fig. 6 A represents the gel electrophoresis plate behind the PCR, wherein swimming lane 4 and the 5 correspondences eluate of device of independent thermal oxide of using by oneself.The result shows the reversibility combination that nucleic acid seldom or not takes place under used condition, because swimming lane 4 is with 5 corresponding eluates and can not see the band relevant with dna fragmentation in test.For relatively, in swimming lane 2 and 3, can remove and see band.Fig. 6 B shows gel electrophoresis plate, wherein swimming lane 4 and 5 pairs of eluates of using the device of thermal oxide+hydrogen peroxide/sulfuric acid cleaned.In addition, do not see nucleic acid belt---this prompting seldom or is not observed DNA and is combined with the reversibility of bond material.
Fig. 6 A, swimming lane 2 and 3 pairs of eluates of using the device of thermal oxide+plasma etching bond material.There is nucleic acid in the band indication, and the bond material of handling like this success bind nucleic acid.Show among Fig. 6 B that swimming lane 2 and 3 is the eluates from the device of thermal oxide+plasma etching+hydrogen peroxide/sulfuric acid cleaned bond material.Can see, have nucleic acid, the reversibility binding events between this indication nucleic acid analyte and the bond material.
Therefore, apparent, according to the present invention, without further processing, the bond material of thermal oxide manufacturing is not enough to the bond material as the segmental DNA of 200 base pairs of investigation in this test separately.Cement Composite Treated by Plasma has shown it is the suitable processing that is used for the bond material of heat of activation oxide compound manufacturing.
Embodiment 3
Present embodiment is used to illustrate the influence of temperature to elution efficiency.
Experiment is carried out on the square silicon with thermal oxidation silicon of 1cm * 1cm.Oxide surface is by using CHF
3And O
2Carry out CHF
3Plasma etch process.5 μ g pure dnas are diluted in the 8 μ l 6M guanidine hydrochloride solutions.Then DNA is placed silicon chip surface, second silicon chip placed on the top, form sandwich and arrange.Then small pieces are placed the gas tight container of controlling moisture to be incubated 15 minutes.Rinsing small pieces three times (each 100 μ l) in fresh Guanidinium hydrochloride are used 70% ethanol rinsing three times (each 100 μ l) subsequently then, make sample in drying at room temperature then, carry out wash-out thereupon.Wash-out wafer four times (amounting to 280 μ l) is used the fresh 10 * TE damping fluid (as described above) of 70 μ l at every turn, and each 5 minutes, under the controlled temperature between 4-80 ℃, as shown in Figure 7.The amount of eluted dna intercalative dye Picogreen
TMQuantitatively.
With reference to figure 7, column diagram shows how the amount of eluted dna varies with temperature, and shows maximum in the time of about 55 ℃, 65 ℃-80 ℃ stable increasing.For the test of carrying out between 4 ℃-80 ℃, 80 ℃ of maximum elution that are issued to DNA.In general, the temperature increase will cause diffusion to increase and the reduction inter-molecular linkage, and maximum elution efficiency should appear at comparatively high temps.Be not limited to any theory, about 55 ℃ peak is considered to the indication of secondary mechanism among Fig. 7.
The experimental result of carrying out on the above-mentioned silicon chip is applicable to micromechanics association reaction device.Reactor and device substrate are heat-insulating, to allow independently heat operation.Resistance heater and RTD are manufactured on (promptly the centering on the well heater of reactor) on glass on the thermal isolation association reaction device.This causes the temperature at binding substances place and to be independent of substrate by electric control.The embodiment 1 described operation manufacturing of the association reaction device of micromachined.Reactor can be 80 ℃ of operations, and substrate is connected with the rest part of guaranteeing system in ambient operation with heat sink, or operates under the temperature that is independent of the binding substances temperature.
The bond material that present embodiment is used to illustrate silane-deposited through or without the joint efficiency of while during Cement Composite Treated by Plasma.These results (being joint efficiency) are further compared with the joint efficiency of thermal oxide+Cement Composite Treated by Plasma bond material, and with all these functions as three temperature.
Experiment is carried out in square the having on thermal oxidation silicon and the silicon based on the PECVD silicon oxide of silane of 1cm * 1cm, deposits at 400 ℃.The thermal oxidation silicon surface is by using CHF
3And O
2Carry out CHF
3Plasma etch process is divided into two groups based on the PECVD oxide compound sample of silane, and wherein one group is also stood CHF
3And O
2Etch processes.5 μ g pure dnas are diluted in the 8 μ l 6M guanidine hydrochloride solutions.Then DNA is placed silicon chip surface, second silicon chip (having identical oxide type with first) placed on the top, form sandwich and arrange.Then small pieces are placed the gas tight container of controlling moisture and under differing temps, be incubated 15 minutes.Rinsing small pieces three times (each 100 μ l) in fresh Guanidinium hydrochloride are used 70% ethanol rinsing three times (each 100 μ l) subsequently then.Make sample in drying at room temperature then, carry out wash-out thereupon.Wash-out wafer three times (amounting to 210 μ l) is used the fresh 10 * TE damping fluid (as described above) of 70 μ l at every turn.Wash-out carried out 20 minutes for the first time, and 15 minutes for the second time, 5 minutes for the third time.All wash-outs all carry out in room temperature.The amount of eluted dna intercalative dye Picogreen
TMQuantitatively.
With reference to figure 8, wherein the x-axle be in degree centigrade in conjunction with temperature, the y-axle is nanogram (ng) number of eluted dna.The result shows the DNA amount (ng) of three kinds of differences in conjunction with wash-out under the temperature.For thermal oxidation silicon with without the PECVD oxide compound based on silane of plasma etch process, maximum combined efficient is 4 ℃ of realizations.All low under all conditions with through plasma etching based on combining of the PECVD oxide compound of silane, and do not strengthen with the change in conjunction with temperature.By cooling and the hot linked heat sink and cooling system substrate to 4 of precipitation ℃, thereby these results are introduced the binding substances of micromachined.
All patents and publication that this paper quotes are incorporated into herein through quoting.Some said structure, function and the operation that is appreciated that above-mentioned embodiment is not that enforcement is essential to the invention, only is to be included in the specification sheets for the integrity of exemplary.In addition, be appreciated that concrete structure, function and the operation of above-mentioned referenced patents and publication can combine and implement with the present invention, but they not crucial for implementing.Therefore should be appreciated that, can beyond specifically describing, implement the present invention, and in fact not depart from the spirit and scope of the invention that claims limit.
Claims (28)
1. be used for the device that miniflow nucleic acid is handled, comprise:
A) silicon substrate;
B) can provide at least one outlet of introducing the microliter amount material, described material is selected from mammalian blood, buffer salt solution, cracking agent, salt brine solution, alcohol, air and combination thereof;
C) mixing tank is positioned at mixing section, and blood can be mixed with buffer salt solution;
D) cracking room can be to wherein sending cracking agent with the lysing cell film;
E) can from device, take out at least one outlet of material;
F) binding substances chamber, comprising can be at the bond material of bind nucleic acid under the conditions suitable: and
G) can instruct stream at least one valve by device.
2. the device of claim 1 also comprises strainer, and it is positioned at the filtration chamber and has enough greatly to catch white corpuscle, enough little aperture to allow red corpuscle to pass through.
3. the device of claim 1, wherein said device design and textural be all-in-one-piece.
4. the device of claim 1, wherein said mixing section is identical with cracking room.
5. the device of claim 2, wherein said cracking room is identical with the filtration chamber.
6. the device of claim 1 also comprises the passage that can pass through at least one optics.
7. the device of claim 1 also comprises the pad that is used to form electrical connection.
8. the device of claim 1 also comprises the chip glass coverture.The device of claim 1 also comprises the heating unit that is used for selectivity heating unit zone.
9. the nucleic acid bond material made of the method through may further comprise the steps:
A) provide silicon substrate;
B) with thermal oxide art breading silicon substrate, so that silicon oxide surface to be provided; With
C) with plasma etching agent plasma in process etching oxidation silicon face.
10. the nucleic acid bond material of claim 10, wherein said plasma etching agent technology comprises CHF
3And O
2The combination of plasma etch process.The nucleic acid bond material that method through may further comprise the steps is made:
A) provide substrate; With
B) will deposit on the substrate based on the silicon oxide of silane with plasma enhanced chemical vapor deposition technology.
11. the nucleic acid bond material of claim 12, wherein said substrate comprises silicon.
12. handle the method for nucleic acid, may further comprise the steps:
A) in mixing section, mix the mammalian blood of microliter amount and the blood mixture that salt brine solution dilutes with generation;
B) make the blood mixture of dilution and cracking agent flow into reaction chamber, wherein said cracking agent makes cell wall rupture and discharges nucleic acid contained in the white corpuscle; With
C) mixture of the nucleic acid that comprises release is flow through in conjunction with the chamber, the wherein said bond material that is included in selective binding nucleic acid under the high content of salt condition in conjunction with the chamber.
13. the method for claim 14 comprises that also the blood mixture that makes described dilution by the filtering step of strainer, separates with red corpuscle with the white corpuscle that will contain nucleic acid.
14. the method for claim 14 also comprises the step with purificant rinsing and described bond material bonded nucleic acid.
15. the method for claim 16, wherein said purificant is an ethanol.
16. the method for claim 14 also comprises step dry with drying means and bond material bonded nucleic acid.
17. the method for claim 18, wherein said drying means is forced convection.
18. the method for claim 18, wherein said drying means comprises airflow.
19. the method for claim 14 also comprises the step with less salt solution-treated and bond material bonded nucleic acid, can collect it so that nucleic acid is free on bond material and makes.
20. be used for the unitary device that miniflow nucleic acid is handled, comprise:
A) with the device of cell material introducing device;
B) with the device of cracking agent introducing device;
C) cell material is mixed to realize the cytolemma cracking and to discharge the device of cellular component with cracking agent;
D) means of selective binding nucleic acid;
E) means of rinsing bonded nucleic acid;
F) means of dry bonded nucleic acid; With
G) go the also means of wash-out nucleic acid of combination.
21. the device of claim 22, wherein said cell material and cracking agent are introduced through inlet.
22. the device of claim 22 wherein makes cell material and cracking agent blended device comprise curling kapillary micro mixer.
23. the device of claim 22, wherein the device of selective binding nucleic acid comprises the bond material of claim 10.
24. the device of claim 22, wherein the device of selective binding nucleic acid comprises the bond material of claim 12.
25. the device of claim 22, wherein the means of selective binding nucleic acid comprise the use high level salt solution.
26. the device of claim 22, the means of wherein dry bonded nucleic acid comprise the forced convection drying.
27. the device of claim 22 wherein goes the means of combination and wash-out nucleic acid to comprise with low salts solution rinsing.
28. the device of claim 22, wherein go in conjunction with and the means of wash-out nucleic acid comprise and use the high purity water rinsing.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US53329703P | 2003-12-30 | 2003-12-30 | |
| US60/533,297 | 2003-12-30 | ||
| US10/818,532 | 2004-04-05 |
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| CN1950506A true CN1950506A (en) | 2007-04-18 |
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ID=38019356
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| CN 200480039520 Pending CN1950506A (en) | 2003-12-30 | 2004-12-27 | Nucleic acid purification chip |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102159333A (en) * | 2008-09-17 | 2011-08-17 | 恰根有限公司 | Method for normalizing contents of biomolecules in sample |
| CN102115711B (en) * | 2010-01-06 | 2013-11-06 | 深圳先进技术研究院 | Micro-flow control chip and nucleic acid extracting and purifying method |
| CN106769693A (en) * | 2016-11-14 | 2017-05-31 | 中国科学院重庆绿色智能技术研究院 | A kind of circulating tumor cell automatic checkout system based on Raman spectrum |
| CN107413401A (en) * | 2010-11-19 | 2017-12-01 | 加利福尼亚大学董事会 | Mixed type flat surface optofluidic integrates |
| CN108085314A (en) * | 2016-11-21 | 2018-05-29 | 清华大学 | A kind of amination filter paper/film purified for nucleic acid extraction and preparation method and application |
| CN112566722A (en) * | 2018-05-16 | 2021-03-26 | 米尔登多微流体系统有限公司 | Microfluidic device and method for separating, purifying and concentrating components of a fluid medium |
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2004
- 2004-12-27 CN CN 200480039520 patent/CN1950506A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102159333A (en) * | 2008-09-17 | 2011-08-17 | 恰根有限公司 | Method for normalizing contents of biomolecules in sample |
| CN102115711B (en) * | 2010-01-06 | 2013-11-06 | 深圳先进技术研究院 | Micro-flow control chip and nucleic acid extracting and purifying method |
| CN107413401A (en) * | 2010-11-19 | 2017-12-01 | 加利福尼亚大学董事会 | Mixed type flat surface optofluidic integrates |
| CN106769693A (en) * | 2016-11-14 | 2017-05-31 | 中国科学院重庆绿色智能技术研究院 | A kind of circulating tumor cell automatic checkout system based on Raman spectrum |
| CN108085314A (en) * | 2016-11-21 | 2018-05-29 | 清华大学 | A kind of amination filter paper/film purified for nucleic acid extraction and preparation method and application |
| CN108085314B (en) * | 2016-11-21 | 2021-11-09 | 杭州梓晶生物有限公司 | Aminated filter paper/membrane for nucleic acid extraction and purification and preparation method and application thereof |
| CN112566722A (en) * | 2018-05-16 | 2021-03-26 | 米尔登多微流体系统有限公司 | Microfluidic device and method for separating, purifying and concentrating components of a fluid medium |
| CN112566722B (en) * | 2018-05-16 | 2022-09-06 | 米尔登多微流体系统有限公司 | Microfluidic device and method for separating, purifying and concentrating components of a fluid medium |
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