CN1379857A - Method and apparatus for stretching polymers - Google Patents
Method and apparatus for stretching polymers Download PDFInfo
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- CN1379857A CN1379857A CN00814266A CN00814266A CN1379857A CN 1379857 A CN1379857 A CN 1379857A CN 00814266 A CN00814266 A CN 00814266A CN 00814266 A CN00814266 A CN 00814266A CN 1379857 A CN1379857 A CN 1379857A
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Abstract
The present invention provides structures and methods that allow polymers of any length, including nucleic acids containing entire genomes, to be stretched into a long, linear conformation for further analysis. The present invention also provides structures and methods for selecting and stretching polymers based on their lengths. Polymers are loaded into a device and run through the structures. Stretching is achieved by, e.g., applying shear forces as the polymer passes through the structures, placing obstacles in the path of the polymer, or a combination thereof. Since multiple molecules may be stretched in succession, extremely high throughput screening, e.g., screening of more than one molecule per second, is achieved.
Description
The application requires the interests of the U.S. Provisional Application 60/149,020 that proposed on August 13rd, 1999, and it draws at this in full and is reference.
1. invention field
The present invention relates to the general field of polymer characterization.More specifically, the present invention relates to utilize structure stretching polymers or be that selective polymer is come on the basis in micromodule with length.
2. background of invention
The diversity of big molecule and biosystem is relevant with the function of essence.Explanation function, dynamics and macromolecular interactional ability depend on the understanding to their chemistry and three-dimensional structure.These three aspect-chemistry and three-dimensional structure and dynamic perfromance-be to be mutually related.For example, the chemical composition of protein and more specifically amino acid whose linear array have determined three-dimensional structure (the Kim ﹠amp that polypeptied chain is folded to form after biosynthesizing clearly; Baldwin (1990) Ann.Rev.Biochem.59:631-660), itself so that determined protein and other macromolecular interactions, and make the mobile relatively of the correct movable phase of protein.
Biomacromolecule be polymkeric substance also be the compound of polymkeric substance.Different types of big molecule is made up of different types of monomer, is 20 seed amino acids with regard to protein promptly, and is four kinds of main nuclear bases (nucleobase) with regard to nucleic acid.A large amount of information can be from determine polymer chain obtains in the line style of monomer or the elementary sequence.For example,, can determine elementary sequence, be used for determining of mRNA expression-form, determine the gene mutation that protein expression form and understanding are corresponding with morbid state so that produce the expression figure by the protein of nucleic acid coding by measuring the elementary sequence of nucleic acid.In addition, distinctive nuclear base sequence can be used for discerning clearly DNA along the distribution characteristics figure of concrete DNA polymkeric substance, as is used for legal medical expert's calibrating.For this reason, quick, accurate and cheap characterize polymers is the method for nucleic acid especially, owing to the effort of the Human Genome Project to the human genome ordering develops.
Characterize the challenge of the linear order of monomer in polymer chain, in most medium, take natural tendency unpredictable, the form of reeling from polymkeric substance.The average of this coiling depends on polymkeric substance and interaction, the rigidity of this polymkeric substance and the energy of polymkeric substance and self-interaction of solution on every side.In most of the cases, this coiling is quite tangible.For example, lambda phage DNA, 16 μ m are long when this DNA of stretching, extension is the B form in theory, have the random coil diameter (Smith etc. (1989) Science 243:203-206) of about 1 μ m.
DNA and many other XC polymer can be modeled as the elasticity rods of the homogeneous of chain twist, to determine their random coil character (Austin etc. (1997) PhysicsToday 50 (2): 32-38).A relevant parameter is a persistence length, P, and directivity is kept on this length, and it is provided by following formula:
P=к/k
BT (1) wherein к is elastic bending modulus (Houseal etc. (1989) Biophys.J.56:507-516), k
BBe that Boltzmann constant and T are temperature (Austin etc. (1997) Physics Today 50 (2): 32-38).The long persistence length meaning refer to polymkeric substance be more rigidity and more unfold.Under physiological condition, for DNA, P ≈ 50nm.Though the molecular diameter than 2.5nm is big, this persistence length is than the general dna molecule little many orders of magnitude of physical length of human chromosome for example, and it is that about 50mm is long.From this persistence length can by following calculated population ball of string size R (Austin etc. (1997) Physics Today 50 (2): 32-38):
＜R
2〉=2PL (2) wherein L is the identical length of dna molecular.With regard to chromosomal DNA, R ≈ 70 μ m.Clearly, more easy on than DNA fragment at resolving information on the long DNA fragment of the 5cm that unfolds with 70 μ m ball of string sizes.
The stretching polymers for example necessary power of DNA is not very big.The spirality chain model makes polymkeric substance be considered to resemble spring, and with its stretch power (Fs) that the natural length completely approach it needs can according to following calculating (Austin etc. (1997) Physics Today 50 (2): 32-38):
F
s≈ k
BWherein all parameter-definitions are as above for T/P (3).Be lower than Fs, the relation between the power that applies and the amount of stretching, extension is roughly linearity; Be higher than Fs, apply bigger power and cause little stretching, extension to change (Smith etc. (1992) Science 258:1122-1126; Bustamante (1994) Science 265:1599-1600).Therefore, stretching completely must be by applying F
sReach.With regard to DNA, it is stretched over the needed power of total length from ball of string form, this extended conformation keeps the B form, is about 0.1pN.So little power can obtain from what source is the fact take up an official post in principle, comprises shearing force, electric power and gravity.
When stretching DNA, danger is not the destruction from covalent bond, and it needs the power of 1nN (Grandbois etc. (1999) Science 283:1727-1730) at least, but is hyper-extended.Have been noted that when applying the power of 70pN DNA adopts super-relaxed shape, so-called " S-DNA " has length (Austin etc. (1997) the Physics Today 50 (2): 32-38) of almost twice in the normal B-form DNA with identical base logarithm.Other people have reported (the Marko ﹠amp of this transformation under 50pN power; Siggia (1995) Macromolecules 28:8759-8770).The length of S-DNA has less consistance than the length of the b form dna that is stretched over its natural length, and more depend on the accurate power (Cluzel etc. (1996) Science 271:792-794) that applies, along with compelling force linearly from 1.7 to 2.1 times to the length variations of b form dna.Because may not know the accurate power that applies, therefore wishing to avoid DNA stretched becomes its S-form.Therefore, have about two number order magnitude range, the power from about 0.1pN to 25pN, can be as one man with predictably stretch DNA and become wide-spread B-form DNA.
In addition, this power must enough apply apace, so that polymkeric substance does not twine again.In the natural relaxation time of polymkeric substance, τ depends on solvent (Marko (1998) Physical ReviewE27:2134-2149), and is as follows:
τ ≈ L
2P μ/k
BT (4) wherein μ is the viscosity of solvent, and other parameters as above define.With regard to the DNA under the physiological condition, this relaxation time is about 6 seconds, in the solution of viscosity, can be increased to 20 seconds (Smith etc. (1999) Science 283:1724-1727) with 220cp, or by making DNA in restriceted envelope, pass through to elongate P and change viscous resistance (Bakajin etc. (1998) Phys.Rev.Let.80:2737-2740).Relaxation time also is the function (Hatfield﹠amp of extension degree; Quake (1999) Phys.Rev.Let.82:3548-3551), so above-mentioned calculated value is the lower limit in the relaxation time of reality.
No matter the exact value in this relaxation time, polymkeric substance must stretch in short time range.Flowing through under the situation of passage, wherein stretching the fluid strain that originates from polymkeric substance, suitable stretching, extension time range is the inverse of strain rate.This strain rate is defined as d ε/dt=dv
x/ dx, wherein x flows to and v
xBe the component of speed along x.Many strain rate and relaxation times are called as Deborah number, De=τ d ε/dt, and can be used for determining whether that stretching, extension will be held (Smith ﹠amp; Chu (1998) Science 281:1335-1340).If De is more many greatly than 1, then adaptability to changes is occupied an leading position and this polymkeric substance will keep extended configuration.If De is little more many than 1, then natural relaxation process prevails and polymkeric substance can not keep extended configuration.When comprising other tensile forces, can obtain dimensionless number by other rational time ranges, for example the Weissenberg number (Smith etc. (1999) Science283:1724-1727) in elongation is flowed.
The prior art that is used to stretch DNA is included at least one end that fixed member is gone up on a surface, controls the other end then, utilizes physical force to stretch, and is fixing then, or by a gel with limiting demensions.Early stage stretching, extension DNA is used for the trial of dimensional measurement to be undertaken by Houseal etc.(1989，Biophys.J.56：507-516)。Dna solution contacted with gold surface produced satisfied bonding, use the Kleinschmidt method, it is widely used in electron microscope technique in order to stretching dna molecular on monolayer of protein, causes many molecules to keep reeling but not stretches.The another kind of trial is by use pipettor " little by little " it to be smeared with stretching, extension DNA, but this technology is difficult to robotization (PCT publication No.WO93/22463).
Design more complicated scheme and be used for an end of fixed dna and other polymkeric substance from the teeth outwards.Usually, they comprise and the surface are exposed to reactive group for example hydroxyl, amine, mercaptan, aldehyde, ketone or carboxyl or add coupling structure such as avidin, streptavidin and biotin with modified surface.The example of these technology is found in PCT publication No.97/06278; U.S. patent No.5,846,724 and Zimmermann ﹠amp; Cox (1994) Nucl.Acids Res.22:492-497.Usually these technology comprise use silane (Bensimon etc. (1994) Science 265:2096-2098).
In case an end of polymkeric substance is fixed, can stretch, because power can be perpendicular to the attaching surface orientation.A commonsense method is to use the receding meniscus arranged polymeric, and this method is sometimes referred to as " molecule combing ".Introduce second fluid in this technology, itself and first fluid are immiscible in fact, form meniscus at described interface.Then by mechanics, heating power, electricity or chemical method or by evaporation first fluid is little by little removed simply and substituted by described new fluid.Therefore when described interface moved, polymkeric substance was aligned to perpendicular to described interface by means of surface tension, became stretching, extension.The stretching force of this method can be expressed as polymkeric substance diameter D (for double-stranded DNA, D=2.2nm) and the function (Bensimon etc. (1994) Science 265:2096-2098) of surface tension γ: F=γ π D.
For the air/water interface, γ is 0.07N/m, and DNA is produced the power of about 40pN, and obviously it is in the scope of requirement.If second fluid is correctly selected to stop polymer motion, then described polymkeric substance is maintained fixed in place in over a long time.In addition, the adjacent polymkeric substance that is attached to similar face is all arranged with equidirectional.Two kinds of related fluids are though the solvent of described polymkeric substance normally can be a partial solvent and a kind of even can be air.Degree of drawing depends on modification (Bensimon, D. etc. (1995) Phys.Rev.Lett.74 (23): 4754-4747), but it is consistent for any given surface treatment on described surface.Used variant (the U.S. patent 5,851,769 of this technology; PCT publication WO97/06278; Bensimon etc. (1994) Science 265:2096-2098; U.S. patent 5,840, and 862; Cox ﹠amp; Zimmermann (1994) Nucl.Acids Res.22:492-497).Yet this technology is not easy to the operation of height-throughput because described fixedly be a kind of rate-limiting step, and be difficult more in described further polymer modification after fixing.
The another kind of method of handling the fixing DNA of an end relates to utilizes ligh trap (opticaltrap).In this technology, by the emission light quantum, laser beam (" light tweezers ") gives dna molecular with momentum.By moving the position of described light quantum, i.e. mobile beam can extremely accurately change the moving direction (U.S. patent 5,079,169, Chu (1991) Science253:861-866) of DNA.Therefore, dna molecular can make with the light tweezers and stretch.The advantage of this technology is to change the power that is used to stretch, and has been used to verify surface creep theory (Perkins etc. (1994) Science 264:819-822).Yet laser can only keep a molecule in position at every turn, and must rearrange for each molecule subsequently, and this makes it not attractive in height-throughput is analyzed.
The third method that stretches DNA relates to the electrophoresis of the DNA that an end is fixed, make molecule independently an end move to direction away from stiff end, and with avidin stiff end is attached to the surface subsequently, perhaps relate to the two ends electrophoresis of DNA freely, with avidin two ends are attached to surface (Kabata etc. (1993) Science 262:1561-1563 then; Zimmerman ﹠amp; Cox (1994) Nucl.Acids Res.22:492-497).Still there is not trial that characterize to use the quality that this technology stretches.In addition, this technology has the shortcoming (with regard to back-fixing means) of above-mentioned technology equally.
Under the situation of fixed member one end not, also DNA is stretched by electrophoresis.As the part that the near field that is used for the sequencing biomolecule is surveyed, DNA extends by electrophoresis in gel and solution, and wherein electrification moves to the appropriate location with DNA and is used for identification (U.S. patent 5,538,898).Yet, do not provide data with definite quality that stretches high molecular polymer, and this technology is limited in analyzing about 3 megabasses at every turn.
The expansion of this thought relates to and utilizes dielectrophoresis, or AC field, to stretch DNA.Washizu and Kurosawa ((1990) IEEE Transactions on IndustryApplications 26:1165-1172) indicate, and can stretch DNA to its total length in the b form dna form in having 106V/m intensity and 400kHz or the field with upper frequency.Under some lower frequency (approximately 10kHz), DNA also can fully stretch, but is perpendicular to the direction of field but not in parallel.This technology has been applied to measure the DNA size, and it has the gap that phases down width by build one between electrode, and DNA is arranged at the length place that gap width equals DNA.Find that also this technology can extending single-stranded DNA, because from the solvent of double-stranded DNA interact different (Washizu etc. (1995) IEEE Transactions onIndustry Applications 31:447-456).A shortcoming of this technology is that owing to have induced dipole along the length of DNA, sample is assembled easily, and is difficult to discern exactly component in heterogeneous sample.In addition, these experiments must be carried out in deionized water, avoiding the effect of undesirable joule of heating and electricity-osmotic flow, thereby bring the specimen preparation difficulty, because most DNA is present in salt solusion or other solvents.
Gravity also is used to stretch DNA (U.S. patent 5,707,797; Windle (1993) Nature Genetics 5:17-21).In this technology, drip from the slide plate that is fixed into certain angle from the DNA of the lauryl sodium sulfate lysing of cell and to flow down.The effect of gravity is enough to stretch DNA, in addition to its excessively-the S-DNA form that stretches.Then this DNA is fixed on the slide plate, carries out for example fluorescent mark before the difficulty stretching relatively.
Church etc. have developed the another kind of method that is used for polymer characterization, and it relates to measurement physical change (U.S. patent 5,795,782) on interface between the two storehouses (pool) of medium when polymkeric substance passes the interface.This method is relatively-stationary.For example, be used for the ion channel scheme (Church etc. (1999) Science 284:1754-1756) that nucleic acid characterizes and be used for single-stranded DNA.The interface that can be used for multiple polymers awaits to develop.
Kambara etc. have developed the assay method (U.S. patent 5,356,776) of DNA length.This method relates to the DNA electrophoresis by gel; When DNA arrived diameter and is not more than several microns gel a part of, it was forced to become straight line, finished fluorescent marker detection on each end of DNA at this.In another embodiment, the end of DNA is fixed in the hole, stretch by electrophoresis, and another terminal mark of molecular detection.Utilize gel to force in the method and use and compare voltage higher in solution, and end mark has been got rid of other signs of great majority of DNA with moving DNA.In addition, the length dna molecule is tending towards tangling in gel.A kind of improved electrophoresis method, i.e. pulse-field electrophoresis (Schwartz ﹠amp; Koval (1989) Nature 338:520-522), can make the longer fragment full extension of DNA by mobile electric field.Yet,, and have other shortcomings of electrophoresis because this technology time spent of change is longer.
Schwartz etc. have developed a kind of be used to stretch the gel base of DNA and the mixed method of solwution method ((1993) Science 262:110-113).DNA is placed in the agarose solution of free fusion, stretches by gravity, is secured in place by the gelification method then.During gelification, also add enzyme, to cut apart DNA in the specificity position.This method is effectively in producing estriction map, yet the predictable stretching, extension in the agarose medium is difficult, and is debatable with the method that this technology is used for height-do not cut DNA by quantitative analysis.
The other technologies that are used to characterize particle do not rely on stretching, extension.For example, method (the U.S. patent 5,599,664 of Schwartz exploitation; EP0391674) come size up and quality by particle being applied power and measurement configuration and change in location.Under the situation of polymkeric substance, this power puts on the form of coiling usually.Another kind is measured the dna molecular size and the method (Chou etc. (1999) Proc.Natl.Acad.Sci.USA 96:11-13) that dna molecular is classified is related to the device of operating at micro-meter scale.This device is used to oneself and analyzes by the integration fluorescence signal of the coiling DNA of wave detector.((1998) Analytical Chemistry70:2303-2310 such as Schmalzing; (1997) Proc.Natl.Acad.Sci. U.S. 94:10273-10278) device of having developed micro-manufacturing is used for DNA analysis, it comprise use conventional art for example electrophoresis the small-scale form and do not rely on the sequencing that DNA stretches.
In order to be determined at the linear order of information in the XC polymer exactly, but must stretch this XC polymer so that the discriminate individuals unit.Though developed the especially technology of DNA of many stretching, extension XC polymer, they have shortcoming, the easiness of consistance of Shen Zhaning and repeatability, processing XC polymer and for example for the applicability of the XC polymer of all types and size.In addition, these methods all are not suitable for the rapid analysis of information, and it is necessary for example in rational hour range the big fragment of DNA being carried out sequencing.Obviously, need a kind of method and apparatus be used for stretching polymers reliably, so that more rapidly and measure the linear order of information wherein exactly, to illustrate complicated hereditary function and to diagnose the illness and hereditary dysfunction.
Should not be construed quoting as proof of this reference is to point out prior art of the present invention.
3. summary of the invention
In first embodiment, the present invention relates to a kind of integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises tapered channel, the width of described tapered channel reduces linearly from first end to the second end, with wherein said at least a polymkeric substance, when existing, move along the direction of described tapered channel from described first end to described second end; Whereby when described at least a polymkeric substance in fluid sample when described tapered channel moves, shearing force is applied on the described at least a polymkeric substance.
This embodiment of the present invention is applicable to stretching polymers, and especially DNA is used for further analysis.
In second embodiment, the present invention relates to a kind of integrating device, it comprises: (a) at least a polymkeric substance in fluid sample; (b) be used to stretch the elongated structure of described at least a polymkeric substance, wherein said elongated structure comprises tapered channel, the width of described tapered channel reduces linearly from first end to the second end, with wherein said at least a polymkeric substance, when existing, move along the direction of described tapered channel from described first end to described second end; Whereby when described at least a polymkeric substance in fluid sample when described tapered channel moves, shearing force is applied on the described at least a polymkeric substance.
In the 3rd embodiment, the present invention relates to a kind of integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises tapered channel, the width of described tapered channel reduces with the speed greater than linearity from first end to the second end, with wherein said at least a polymkeric substance, when existing, move along the direction of described tapered channel from described first end to described second end; Whereby when described at least a polymkeric substance in fluid sample when described tapered channel moves, shearing force is applied on the described at least a polymkeric substance.
This embodiment of the present invention also is applicable to stretching polymers, and especially DNA is used for further analysis.
In the 4th embodiment; The present invention relates to a kind of integrating device; It comprises: (a) at least a polymkeric substance in fluid sample; (b) be used to stretch the elongated structure of described at least a polymkeric substance; Wherein said elongated structure comprises tapered channel; The width of described tapered channel reduces with the speed greater than linearity from first end to the second end; With wherein said at least a polymkeric substance; When existing; Move along the direction of described tapered channel from described first end to described second end; Whereby when described at least a polymkeric substance in fluid sample when described tapered channel moves; Shearing force is applied on the described at least a polymkeric substance.
In the 5th embodiment, the present invention relates to a kind of integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises tapered channel, the width of described tapered channel reduces from first end to the second end, with wherein said at least a polymkeric substance, when existing, move along the direction of described tapered channel from described first end to described second end; Whereby when described at least a polymkeric substance in fluid sample when described tapered channel moves, shearing force is applied on the described at least a polymkeric substance, wherein said shearing force produces constant shear rate.
This embodiment of the present invention is applicable to stretching polymers, and especially DNA is used for further analysis.
In the 6th embodiment, the present invention relates to a kind of integrating device, it comprises: (a) at least a polymkeric substance in fluid sample; (b) be used to stretch the elongated structure of described at least a polymkeric substance, wherein said elongated structure comprises tapered channel, the width of described tapered channel reduces from first end to the second end, with wherein said at least a polymkeric substance, when existing, move along the direction of described tapered channel from described first end to described second end; Whereby when described at least a polymkeric substance in fluid sample when described tapered channel moves, shearing force is applied on the described at least a polymkeric substance, wherein said shearing force produces constant shear rate.
In the 7th embodiment, the present invention relates to a kind of integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises elongated structure, and wherein said elongated structure comprises a centre gangway that is used to hold fluid and manyly is used to hold the lateral access that fluid is connected to described centre gangway; With wherein said at least a polymkeric substance, when existing, on prolonging direction, move along described centre gangway.
This embodiment of the present invention is applicable to stretching polymers, and especially DNA is used for further analysis.
In the 8th embodiment, the present invention relates to a kind of integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises: (a) a kind of elongated structure; (b) a kind of transfer passage, its introducing and draw described elongated structure is used for carrying described at least a polymer samples at described fluid to described elongated structure; (c) be used for making described at least a polymkeric substance at described fluid sample, when existing, the equipment that moves in described elongated structure, wherein said elongated structure comprise a centre gangway that is used to hold fluid and manyly are used to hold the lateral access that fluid is connected to described centre gangway; Wherein when described at least a polymkeric substance existed, described equipment made described at least a polymkeric substance move on prolonging direction along described centre gangway.
This embodiment of the present invention is applicable to stretching polymers, and especially DNA is used for further analysis.
In the 9th embodiment, the present invention relates to a kind of integrating device that is used for extending in the DNA of fluid sample, it comprises: (a) a kind of elongated structure; (b) be used for the described DNA that carries at described fluid sample equipment to described elongated structure; (c) be used for the described DNA that makes at described fluid sample, when existing, the equipment that moves in described elongated structure, wherein said elongated structure comprise a centre gangway that is used to hold fluid and manyly are used to hold the lateral access that fluid is connected to described centre gangway; Wherein when described DNA existed, described equipment made described DNA move on prolonging direction along described centre gangway.
In the tenth embodiment, the present invention relates to a kind of integrating device, it comprises: (a) at least a polymkeric substance in fluid sample; (b) be used to stretch the elongated structure of described at least a polymkeric substance, wherein said elongated structure comprises a centre gangway that is used to hold fluid and manyly is used to hold the lateral access that fluid is connected to described centre gangway.
In the 11 embodiment, the present invention relates to a kind of integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises that has at least one crooked passage, with wherein said at least a polymkeric substance, when existing, move along described passage.
This embodiment of the present invention is applicable to stretching polymers, and especially DNA is used for further analysis.
In the 12 embodiment, the present invention relates to a kind of integrating device that is used for extending in the DNA of fluid sample, it comprises: (a) a kind of elongated structure; (b) be used for carrying the equipment of described DNA at described fluid sample to described elongated structure, wherein said elongated structure comprises that one has at least one crooked passage and wherein said DNA, when existing, moves along described passage.
In the 13 embodiment, the present invention relates to a kind of integrating device, it comprises: (a) at least a polymkeric substance in fluid sample; (b) a kind of elongated structure that is used to stretch described at least a polymkeric substance, wherein said elongated structure comprise that has at least one crooked passage.
In the 14 embodiment, the present invention relates to a kind of integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises a tapered channel, along the described at least a polymkeric substance of this tapered channel, when existing, move and wherein said passage comprises the barrier of many described at least a polymer motion at flow direction.
This embodiment of the present invention is applicable to stretching polymers, and especially DNA is used for further analysis.
In the 15 embodiment, the present invention relates to a kind of integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises a centre gangway, along the described at least a polymkeric substance of this centre gangway, when existing, move at flow direction, with the many lateral access of described centre gangway and moving obstacles that wherein said centre gangway further comprises many described at least a polymkeric substance of being connected to.
In the 16 embodiment, the present invention relates to a kind of integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises that has at least one crooked passage, along the described at least a polymkeric substance of this passage, when existing, move and wherein said passage comprises the moving obstacle of many described at least a polymkeric substance at flow direction.
In the 17 embodiment, the present invention relates to a kind of integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises a passage, along the described at least a polymkeric substance of this passage, when existing, move at flow direction, comprise many pillars with wherein said passage, at least one of described pillar has the polygonal cross sectional shape of non-quadrilateral.
The the 15, the 16 and the 17 embodiment of the present invention is applicable to stretching polymers, and especially DNA is used for further analysis.
In the 18 embodiment, the present invention relates to a kind of integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises a passage, along the described at least a polymkeric substance of this passage, when existing, on flow direction, move, comprise the moving obstacle of many described at least a polymkeric substance with wherein said passage, described many barriers are oriented to a series of row, each described registration becomes perpendicular to described flow direction, depart from each continuous row and last row, whereby along described flow direction, at least one is not equal to the part of 1/2 multiple of one of described barrier, the extension in the overlapping gap that is formed by two adjacent barrier in described last row.
This embodiment of the present invention is applicable to stretching polymers, and especially DNA is used for further analysis.
In the nineteen embodiment, the present invention relates to a kind of integrating device, it comprises (a) at least a polymkeric substance in fluid sample, every kind of described polymkeric substance has the diameter more than or equal to minimum diameter; (b) a kind of elongated structure that is used to stretch described at least a polymkeric substance, wherein said elongated structure comprises a passage, along the described at least a polymkeric substance of this passage, when existing, on flow direction, move, comprise the moving obstacle of many described at least a polymkeric substance with wherein said passage, described many barriers are oriented to a series of row, each described row perpendicular to described flow direction location and each the adjacent barrier in each described row's series to separating by 50 times distance greater than described minimum diameter.
This embodiment of the present invention is applicable to stretching polymers, and especially DNA is used for further analysis.
In the 20 embodiment, the present invention relates to a kind of integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises a passage, along the described at least a polymkeric substance of this passage, when existing, on flow direction, move, comprise the moving obstacle of many described at least a polymkeric substance with wherein said passage, described many barriers reduce dimensionally along described flow direction.
This embodiment of the present invention also is applicable to stretching polymers, and especially DNA is used for further analysis.
In the 21 embodiment, the present invention relates to a kind of integrating device that is used to stretch DNA, it comprises a kind of elongated structure, wherein said elongated structure comprises the centre gangway of a taper, the centre gangway of described taper comprises first end and second end, with wherein said DNA, when existing, move in direction along described tapered center passage from described first end to described second end, wherein said elongation further comprises many lateral access that are connected to described tapered center passage, and wherein said tapered center passage comprises at least one bending; Comprise the moving obstacle of many described DNA with wherein said tapered center passage.
In the 22 embodiment, the present invention relates to a kind of integrating device that is used to stretch DNA, it comprises a kind of elongated structure, described elongated structure comprises: (a) one first tapered channel, described first tapered channel comprises one first end, one second end and between described first end and described second end staggered many pillars, it constitutes 12-15 row, the width of described first tapered channel reduces with 26.6 ° angle, described angle at described first end with respect to a constant-width channel definition, described first end has the width between 0.5 and 5 μ m, and described pillar has and equals 1.5 μ m
2Cross-sectional area and the gap that is equaled 0.5 μ m separate; (b) one second tapered channel, described second tapered channel is connected to described first tapered channel and width is decreased between the 0.5 and 5 μ m at described second end, therefore a shearing force that produces constant shear rate is applied to described DNA, when it exists, described second tapered channel have 1 and 3mm between length.
In the 23 embodiment, the present invention relates to be used to stretch the method for at least a polymkeric substance, it may further comprise the steps: (a) carry described at least a polymkeric substance to a kind of elongated structure, described elongated structure comprises a tapered channel with first end and second end; (b) move described at least a polymkeric substance to described second end along described tapered channel from described first end, described whereby tapered channel makes a shearing force that produces constant shear rate,, when moving, described tapered channel is applied on the described at least a polymkeric substance at described at least a polymkeric substance.
The method that this embodiment of the present invention comprises is applicable to stretching polymers, and especially DNA is used for further analysis.
In the 24 embodiment, the present invention relates to a kind of method that is used to stretch at least a polymkeric substance, it may further comprise the steps: (a) carry described at least a polymkeric substance to a kind of elongated structure, described elongated structure comprises a passage with convergent linearly of one first end and one second end; (b) move described at least a polymkeric substance from described first end to described second end along described tapered channel.
In the 25 embodiment, the present invention relates to a kind of method that is used to stretch at least a polymkeric substance, it may further comprise the steps: (a) carry described at least a polymkeric substance to a kind of elongated structure, described elongated structure comprises a tapered channel with one first end and one second end, and described tapered channel dwindles from described first end to described second end with the speed greater than linearity; (b) move described at least a polymkeric substance from described first end to described second end along described tapered channel.
In the 26 embodiment, the present invention relates to a kind of method that is used to stretch at least a polymkeric substance, it may further comprise the steps: (a) carry described at least a polymkeric substance to a kind of elongated structure, described elongated structure comprises that one is held the centre gangway of fluid and many fluids that holds are connected to described centre gangway lateral access, and described centre gangway comprises one first end and one second end; (b) move described at least a polymkeric substance from described first end to described second end along described centre gangway.
The of the present invention the 24, the 25 and the method for the 26 embodiment be applicable to stretching polymers, especially DNA is used for further analysis.
In the 27 embodiment, the present invention relates to a kind of method that is used to stretch at least a polymkeric substance, it may further comprise the steps: (a) carry described at least a polymkeric substance to a kind of elongated structure, described elongated structure comprises that has at least one crooked passage, and described passage comprises one first end and one second end; (b) move described at least a polymkeric substance from described first end to described second end along described passage.
The method that this embodiment of the present invention comprises is applicable to stretching polymers, and especially DNA is used for further analysis.
In the 28 embodiment, the present invention relates to a kind of method that is used to stretch at least a polymkeric substance, it may further comprise the steps: (a) carry described at least a polymkeric substance to a kind of elongated structure, described elongated structure comprises the moving obstacle of a passage and the many described at least a polymkeric substance in described passage, and described centre gangway comprises one first end and one second end; (b) move described at least a polymkeric substance along described passage from described first end to described second end, wherein said many moving obstacles reduce dimensionally along the direction from described first end to described second end.
The method that this embodiment of the present invention comprises is applicable to stretching polymers, and especially DNA is used for further analysis.
In the second nineteen embodiment, the present invention relates to a kind of method that is used to stretch at least a polymkeric substance, it may further comprise the steps: (a) carry described at least a polymkeric substance to a kind of elongated structure, described elongated structure comprises the moving obstacle of a passage and the many described at least a polymkeric substance in described passage, and described centre gangway comprises one first end and one second end; (b) move described at least a polymkeric substance from described first end to described second end along described passage, at least one of wherein said barrier has non-tetragonal polygon shape of cross section.
The method that this embodiment of the present invention comprises is applicable to stretching polymers, and especially DNA is used for further analysis.
In the 30 embodiment, the present invention relates to a kind of method that is used to stretch at least a polymkeric substance, it may further comprise the steps: (a) carry described at least a polymkeric substance to a kind of elongated structure, described elongated structure comprises: (i) one has at least one crooked tapered center passage, and described tapered center passage comprises one first end and one second end; (ii) many lateral access that is connected to described tapered center passage; The (iii) moving obstacle of the many described at least a polymkeric substance in described tapered center passage; (b) move described at least a polymkeric substance from described first end to described second end along described centre gangway.
The method that this embodiment of the present invention comprises is applicable to stretching polymers, and especially DNA is used for further analysis.
In the 31 embodiment, the present invention relates to be used for to stretch the integrating device of at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, and wherein said elongated structure comprises a passage, along the described at least a polymkeric substance of this passage, when existing, move on flow direction and wherein said passage comprises at least one step, it makes the height of described passage, z reduces to second end from first end.
In the 32 embodiment, the present invention relates to a kind of integrating device, it comprises a kind of elongated structure that comprises a passage, described passage comprises at least one step, and it makes the height of described passage, z, reduce from first end to the second end, described passage comprises at least a polymkeric substance in fluid sample, and being arranged so that at described at least a polymkeric substance of described passage when mobile, applying a shearing force thereon from described first end on the direction of described second end.
In the 33 embodiment, the present invention relates to a kind of integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, and described elongated structure comprises: (a) first passage, described first passage comprise one first end and one second end; (b) second channel, described second channel comprises one the 3rd end and one the 4th end, described the 3rd end is connected to described first passage at described second end, along the described at least a polymkeric substance of passage, when existing, move on flow direction and wherein said first passage reduces at width from described first end to described second end, its speed that reduces is different from the speed that described second channel reduces at width to described the 4th end from described the 3rd end.
In the 34 embodiment, the present invention relates to a kind of integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, described elongated structure comprises: (a) first passage, its width equals 10 μ m and highly equals 1 μ m, described first passage comprises one first end, second end and staggered many pillars between described first end and described second end, it constitutes at least 12 to 15 rows, described many pillars finish at described second end, and each has 1-25 μ m in described many pillars
2Cross-sectional area; (b) second channel, described second channel comprise one the 3rd end and one the 4th end, and described the 3rd end is connected to described first passage at described second end, described second channel from described the 3rd end to described the 4th end with 1/x
2Speed dwindle, described beam overall narrows down to 1 μ m from 10 μ m, wherein x is the distance along the length of described second channel, the length of described second channel equals 5 μ m, described second channel is included in height to the 0.25 μ m that described the 3rd end reduces described second channel
2A step, wherein said at least a polymkeric substance when existing, moves on flow direction along described first passage and described second channel.
In the 35 embodiment, the present invention relates to a kind of being used for length is the integrating device that at least a polymkeric substance at fluid sample is stretched on the basis selectively, it comprises a kind of elongated structure, wherein said elongated structure comprises: (a) first passage, described first passage comprise one first end, second end and between described first end and described second end staggered many pillars, each pillar is located in described second end of distance and is not less than L in described many pillars; (b) second channel, described second channel comprises one the 3rd end and one the 4th end, described the 3rd end is connected to described first passage at described second end, described second channel dwindles at width to described the 4th end from described the 3rd end, along the described at least a polymkeric substance of passage, when existing, on flow direction, move.
In the 36 embodiment, the present invention relates to a kind of many integrating devices that are used for stretching at the polymkeric substance of fluid sample with different length, it comprises a kind of elongated structure, wherein said elongated structure comprises: (a) first passage, described first passage comprise one first end and one second end; (b) second channel, described second channel comprise one the 3rd end and one the 4th end, and described the 3rd end is connected to described first passage at described second end, and described second channel dwindles at width to described the 4th end from described the 3rd end; (c) staggered many pillars in described first passage and described second channel along the described many polymkeric substance of passage, when existing, move on flow direction.
In the 37 embodiment, the present invention relates to a kind of method that is used to stretch at least a polymkeric substance, it comprises along a kind of elongated structure and moves described at least a polymkeric substance, described elongated structure comprises a first passage, and described first passage comprises one first end and one second end; With a second channel, described second channel comprises one the 3rd end and one the 4th end, described the 3rd end is connected to described first passage at described second end, wherein said first passage reduces at width from described first end to described second end, and its speed that reduces is different from the speed that described second channel reduces at width to described the 4th end from described the 3rd end.
In the 38 embodiment, the present invention relates to a kind of be used for stretching at least a at fluid sample, length is more than or equal to the method for the polymkeric substance of L, it comprises along a kind of elongated structure and moves described at least a polymkeric substance, described elongated structure comprises a first passage, described first passage comprises one first end, one second end and between described first end and described second end staggered many pillars, it is L that each pillar in described many pillars is located in described second end of distance, with a second channel, described second channel comprises one the 3rd end and one the 4th end, described the 3rd end is connected to described first passage at described second end, described second channel dwindles at width to described the 4th end from described the 3rd end, has wherein that polymkeric substance more than or equal to the length of L stretches and the polymkeric substance that has less than the length of L is not stretched.
In the 3rd nineteen embodiment, the present invention relates to a kind of many methods that are used for stretching at the polymkeric substance of fluid sample with different length, it comprises along a kind of elongated structure and moves described many polymkeric substance, described elongated structure comprises: (a) first passage, described first passage comprise one first end and one second end; (b) second channel, described second channel comprise one the 3rd end and one the 4th end, and described the 3rd end is connected to described first passage at described second end, and described second channel dwindles at width to described the 4th end from described the 3rd end; (c) staggered many pillars in described first passage and described second channel.
In the 40 embodiment, the present invention relates to a kind of method that is used to stretch at least a polymkeric substance, it comprises along a kind of elongated structure and moves described at least a polymkeric substance, described elongated structure comprises: (a) width equals 10 μ m and highly equals the first passage of 1 μ m, described first passage comprises one first end, one second end and between described first end and described second end staggered many pillars, it constitutes at least 12 to 15 rows, described many pillars have 1-25 μ m in described second end end and each pillar in described many pillars
2Cross-sectional area; (b) second channel, described second channel comprise one the 3rd end and one the 4th end, and described the 3rd end is connected to described first passage at described second end, described second channel from described the 3rd end to described the 4th end on width with 1/x
2Speed dwindle, described beam overall is contracted to 1 μ m from 10 μ m, wherein x is the distance along the length of described second channel, the length of described second channel equals 5 μ m, described second channel is included in height to the 0.25 μ m that described the 3rd end reduces described second channel
2Step.
4. accompanying drawing is described
Fig. 1 shows the various example of structure that belong to the scope of the invention.
Fig. 2 (a-m) represents: the several embodiments that (a) comprise the stretched out structure of funnel, pillar, branch and cascaded structure; (b) the amplification example of continuous two-funnel structure with pillar; (c) several embodiments of compound pillar arrangement and branched structure; (d) comprise the embodiment of the series connection and the structure of parallel-connection structure; (e) asymmetric branched structure; (f) has the structure of the little barrier combination that limits little gap; (g) has the structure of the combination of polygon, grid and pillar; (h) asymmetric bending structure; (i) has the enlarged drawing of the branched structure of pillar; (j) has the big funnel structure of pillar; (k) has the funnel structure of pillar; (l) has the linear funnel structure that increases flow velocity, has and do not have pillar; The summary of some funnel structure that (m) the present invention includes.
Fig. 3 illustrates the embodiment of shearing-stretching, extension mode of using the constant tapered channels.
Fig. 4 explanation is wherein in the embodiment of shearing-stretching, extension mode that shear rate increases rapidly when passage length carries out that flows.
Fig. 5 illustrates the embodiment of the shearing-stretching, extension mode of the tapered channel that is used for producing constant shearing force.
The embodiment from the shearing-stretching, extension mode of the fluid of passage adding is from the side wherein sheared in Fig. 6 explanation.
Fig. 7 (a) shows how shearing force produces, and wherein Ju Bu rotation and stretching component of force are almost equal in slype; (b) show how shearing force produces when adding fluid generation power, and wherein tensile force surpasses revolving force.
Fig. 8 explanation is wherein sheared from passage and the embodiment that has the shearing-stretching, extension mode of lateral access of narrowing down.
Fig. 9 (a) illustrates fluid " runway effect ", in the fluid of curved outside and comparing by the longer time of bend usefulness in the inboard; (b) show how " runway effect " causes the stretching, extension of polymkeric substance in bending.
Figure 10 shows the embodiment of tortuous mode, and wherein passage is sine wave shape.
Figure 11 shows the embodiment of tortuous mode, wherein the shape of passage indention.
Figure 12 shows the embodiment of tortuous mode, and wherein passage is " snake " shape right angle.
Figure 13 shows when polymkeric substance bending channel when passage advances how to be used for multiple detection.
Figure 14 is presented at how polymkeric substance stretches in the embodiment of the barrier district mode with classification barrier size.
Figure 15 illustrates a kind of coordinate frame of elongated structure.
Figure 16 (a) illustrates the embodiment of the barrier district mode of the circular barrier with square-grid; (b) embodiment of the barrier district mode of the circular barrier with dislocation-grid is described.
Figure 17 explanation has the embodiment of the rectangle barrier close interval barrier district mode of amplifying aspect ratio.
Figure 18 explanation has the embodiment of the barrier district mode of circular barrier close interval.
Figure 19 illustrates the embodiment of the barrier district mode of the circular barrier with three classification dimensions.
Figure 20 explanation is used to have the consistent structure of scattering, carrying and stretching of the DNA of different size.
Figure 21 explanation is used to stretch the structure of preferred version of the structure of DNA, its column subarea, with 1/x
2The funnel of gradual change, wherein x is along the distance of the length of funnel and reduces the step of channel height.
Figure 22 illustrates the sketch of molecular dimension screening installation, and wherein length is that the signal of L or bigger molecule can be easily and the signal distinguishing of length less than the molecule of L.
Figure 23 illustrates the device sketch of the molecule that stretches all length, and wherein the signal from all molecules is uniformly detected.
Figure 24 illustrates and uses the confocal fluorescent illumination and the sensitive optical devices of detection.
Figure 25 represents the embodiment of whole polymer analysis system.
Figure 26 explanation is at the various extended configurations of constant-shearing feeder connection DNA of place.
Figure 27 (a-g) illustrates that 50kb DNA is stretched in a tapered channel.
The DNA that Figure 28 explanation is stretched in a passage in the 537kb measurement.
Figure 29 illustrates the histogram that shows with the DNA length of determination of experimental method.
Figure 30 illustrates the histogram with the phage DNA length of determination of experimental method, and it is from no pillar of Figure 20 (a) and the structure that pillar (b) is arranged.
5. detailed Description Of The Invention
5.1 introduction
The invention provides the polymkeric substance with any length, comprise the nucleic acid that comprises the full gene group, stretching becomes long, linear form and is used for the structure further analyzed.Under especially physics, electricity or chemical force promoted, polymkeric substance was transfused to a device and passes described structure.Stretching, extension is by for example applying shearing force during by this structure, place barrier or it is in conjunction with finishing in polymer path when polymkeric substance.Because described power is applied continuously, therefore can stretching polymers to the length of the effective coverage that is equal to or greater than described device, promptly wherein when polymkeric substance is analyzed the information about described polymkeric substance be collected.For example, if the volume of video camera or laser radiation is concentrated on the zone that the micromodule that stretches takes place, then can monitor the dna molecular of indefinite length, promptly than described video image or the big many length of laser radiation volume.Because many molecules can stretch continuously, therefore obtain the extremely screening of high throughput, for example surpass the screening of per second a part.
The polymkeric substance of stretching, extension or the colony of polymkeric substance are characterized.Polymkeric substance that stretch, mark moves through at least one measuring point, and the indexing unit of said polymkeric substance and the interaction of described measuring point produce the pulse to picture-dependence.As employed in this application, " move through " and refer in embodiments, measuring point be fix and the polymkeric substance that stretches is kept in motion, measuring point is kept in motion and the polymkeric substance of described stretching, extension is that fix to be kept in motion with polymkeric substance both described measuring point and stretching, extension.
Though the present invention can be used to characterize any polymkeric substance, preferably described polymkeric substance has the ground of preponderating, though be not to be the arrangement of linearity or strand exclusively.The example of such polymkeric substance comprises XC polymer for example DNA (deoxyribonucleic acid), RNA (ribonucleic acid), polypeptide and compound sugar.Described polymkeric substance can be heterogeneous on main chain is formed, and therefore comprises any possible combination of the individual monomer units that links together, peptide-nucleic acid (PNA) for example, and it has the amino acid that is connected to nucleic acid.In a preferred version, described polymkeric substance be on main chain is formed homogeneous and be for example nucleic acid, polypeptide or compound sugar.Term " main chain " has its meaning common aspect high polymer chemistry.Nucleic acid used herein is a kind of XC polymer, comprises nucleotide, for example DNA (deoxyribonucleic acid) (DNA) or RNA (ribonucleic acid) (RNA).The XC polymer that protein used herein or polypeptide are made up of amino acid.In the most preferred embodiment, the material of extension is a double chain DNA molecule.
With respect to the individual cell of polymkeric substance, " connection " used herein or " connection " refer to that two unit link to each other by any physical chemistry means each other.Comprise the connection known to any those of ordinary skills, covalency or non-covalent.The nature bonding, for example acid amides, ester and thioester bond are prevailing, it is the special polymkeric substance individual cell of connection usually found at occurring in nature those.Yet, can be that bonding that be synthesized or modification connects by the polymkeric substance individual cell of construction stretch of the present invention.
Polymkeric substance is made of many individual cell, and it is building block or monomer, and it directly or indirectly is connected to other building blocks or monomer with the described polymkeric substance of form.Described polymkeric substance preferably includes the monomer of at least two kinds of different connections on chemical property.The monomer of described at least two kinds of different connections on chemical property can produce or be labeled to produce different signals.Different types of polymkeric substance is made up of different monomers.For example, DNA is the XC polymer that comprises deoxyribose phosphate ester main chain, on this main chain, be connected with purine and pyrimidine for example adenine, cytimidine, guanine, thymine, 5-methylcytosine, 2-amino-purine, hypoxantine and other naturally with the non-nuclear base that exists naturally, replacement and unsubstituted aromatics part.RNA is the XC polymer that comprises the ribose phosphate main chain, be connected with purine and pyrimidine for example describe for DNA those on this main chain, but wherein uracil replaces thymidine.Deoxyribonucleotide can by means of by 5 ' or the ester bond of 3 ' hydroxyl be connected to each other with the described DNA polymkeric substance of form.Ribonucleotide can by means of by 5 ', 3 ' or the ester bond of 2 ' hydroxyl be connected to each other.In addition, have 5 ', 3 ' or 2 ' amino DNA or RNA unit other unit that can be connected to polymkeric substance by means of amido link.
Described polymkeric substance can be the polymkeric substance that exists naturally that nature exists or non-.Polymkeric substance can use the biological chemistry purification techniques for example to separate from natural source.In addition, polymkeric substance can be synthesized, for example by using the external amplification of enzymatic PCR (PCR), by chemosynthesis or by the recombinant technology.
The method coupling of structure of the present invention and the polymkeric substance that is used to analyze stretching, extension, described method detects the signal to picture-dependence pulse." to picture-dependence pulse " used herein is a kind of physical quantity that detects, its transmission or express the information of the architectural characteristic of at least one unit of relevant stretching polymers-peculiar mark.A unit-distinctive mark used herein, it can be the inherent characteristic of measurable stretching polymers particular types individual unit, the different absorption maximum of the nuclear base of the DNA (this polymkeric substance is by the intrinsic ground mark) that exists naturally for example, or having a compound of measurable property, one or more individual cell of itself and polymkeric substance (this polymkeric substance is by external ground mark) have specific contact.The unit of the polymkeric substance of external ground mark-distinctive mark can be specific fluorescent dye, and with its whole nuclear bases with particular type, for example the whole thymines in the DNA chain are examined the base marks.In addition, the unit of the polymkeric substance of external ground mark-distinctive mark can be the qualification length of fluorescence mode mark and the oligonucleotide of sequence, its hydridization and therefore " mark " be present in complementary series in the target dna.Unit-distinctive mark may further include, but is not limited to, distinctive main or less important groove bonding agent (groove binder) and the intercalator of sequence, and sequence-distinctive DNA or peptide-binding proteins matter, the PNAs that sequence is specific, or the like.Detectable physical quantity can be the measured any form of energy.For example, detectable physical quantity can be that electromagnetic radiation, chemical electricity are led, radioactivity or the like.Described pulse to picture-dependence can produce from energy shift, control excites, quencher, lead change or any other physical change aspect (resistance) at electricity.In one embodiment, described pulse to picture-dependence produces the fluorescence resonance energy transmission (" FRET ") between comfortable described unit-distinctive mark and described measuring point or the described measuring point environment on every side.In preferred embodiments, use comes from directly exciting in restriction or the locating area to picture-dependence pulse, or the epiillumination that excites of confocal volume or slit-Ji.The possible analysis of polymkeric substance comprises, but be not limited to: the mensuration of polymkeric substance length, the mensuration of polymer sequence, the mensuration of polymer velocity, the mensuration of the homogeneity degree of two kinds of polymkeric substance, the mensuration of the characteristic pattern of polymer unit-distinctive mark is to produce the sign of " fingerprint " and use unit-polymkeric substance heterogeneous population that distinctive tokens statistics distributes in sample population.
Have many methods and product can be used for analyzing polymers, WO98/35012 is described as the PCT publication, and it draws at this in full and is reference.
The whole bag of tricks that is used for analyzing polymers is different aspect their potential sensitivity and resolution, i.e. bee-line between two unit-distinctive mark, and the distinctive mark in wherein said unit is differentiable.The low resolution technology can be distinguished the unit-distinctive mark that has big distance between them; High resolution technique can be distinguished the unit-distinctive mark that has small distance between them.The resolution of particular technology is measured by characteristic distance, can detect the specific unit-peculiar mark of stretching polymers apart from measuring point at this.Short characteristic distance helps The better resolution.Minimum resolution technique comprises that monitoring transmittance and control excite, and its resolution is 50-100nm or above (Tan ﹠amp; Kopelman (1996) Chem.Anal.Ser.137:407-475).By contrast, the resolution of FRET is about F rster radius, is giving the distance that the most effective energy transfer takes place between body and the receptacle, and it is typically 2-7nm.Distance in a dna molecular with B-form that fully stretches between adjacent base-pair is 3.4 or 0.34nm.State of nature in solution, DNA does not exist with the B-form of its full extension, but exists as the ball of string with about 10 μ m diameters.Therefore, be difficult to resolve the many unit-distinctive mark on the dna molecular of a coiling, so this molecule should stretch before analyzing.
5.2 shearing force is as the means of stretching polymers
When polymer molecule ran into the barrier of physics, it will or not have the ground of interaction process, or " hooking " this barrier, makes the part of this chain remain on each side of this barrier.This does not mean that this polymkeric substance is bonded to this barrier or physically adheres on the contrary.The imbalance that hangs around barrier determines the speed that molecule is advanced along favourable side.(referring to Austin ﹠amp; Volkmuth, Analysis 1993 (21) 235-238.) in addition, produce the localization velocity gradient at the barrier place, reduce because can be used for the mobile cross-sectional area of fluid.As a result, fluid flows than before and faster afterwards between barrier.This has produced the shearing force that acts on approaching molecule, and it plays the effect of the tensile force on polymkeric substance.When this effect was had the overall region expansion of the barrier of correctly adjusting size, polymkeric substance stretched to pass through the whole barriers in this zone.In a preferred embodiment, polymkeric substance is stretched with linear mode.
In case when this polymkeric substance enters a passage by this barrier series and in its wide-spread mode, in preferred embodiments it is analyzed herein, it will tend to the form of getting back to lower-energy, reeling more naturally.For preventing its generation, passage is designed to a constant shearing force is provided on this polymkeric substance in the passage that narrows down, and it is remained in the extended conformation.
Constant shear rate, or in passage along with distance is changing aspect the average velocity, be defined as S:
u/ x=S (5) wherein x is a distance along the substantial rectangular passage, and u is at the axial average fluid velocity of x, and its cross-sectional area A by whole liquid flow (Q) and passage is calculated as follows:
u＝Q/A (6)。
In one embodiment, wherein channel cross-section is a rectangle, and passage can be limited by constant altitude H and width W, makes cross-sectional area A=HW, so average fluid velocity is provided by following formula:
U=Q/HW (7) application boundary condition, promptly liquid flow must be continuous (that is, incompressible), then Q is constant.Therefore, u and W are inversely proportional to.The expression that this relation can substitution be used for S at first is with the relation of decision between shear rate and width:
s＝u/x＝Q/H?/x(1/W)＝(-Q/HW
2)(dW/dx) (8)
DW/dx=(SH/Q) (W
2) (9) to this expression formula integration, find:
W=(SHx/Q+C)
-1(10) wherein C is a integration constant by the decision of the initial width (boundary condition) of passage.This equation for channel width is used for limiting pillar structure passage in addition.For the non-rectangle channel shape, those skilled in the art can easily finish similar calculating.When clean momentum transfer not taking place on altitude axis, promptly when when z-axle medium velocity distribution form has formed, shear rate produces tensile force from the width section.Give an example under the Newtonian fluid situation stress tensor τ that computing power needs
YzCan easily represent with shear rate:
F＝∫∫-τ
yzdzdx＝∫∫-μ(du/dx)dzdx＝∫∫-μSdzdx， (11)
Wherein μ is a solution viscosity.In these equations, x is a direction of motion, and y is that width and z are height.The shear rate surface of integration thereon is the conduit wall surface, the result:
F=μ HLS (12) wherein L is the length of conduit wall, is approximately the length that keeps shearing constant passage therein.
Therefore, the aquaporin with 1 μ m height, 1mm length and 0.25/s shear rate produces the power of about 0.25pN, is enough to stretch DNA, and this is confirmed with experimental technique by the inventor.To it should be noted that this result confirms constant-shear the elongation of the DNA that passage not only keeps in advance-stretch, and help further to stretch DNA, or stretch DNA by oneself.
In a preferred embodiment, combine two kinds and be used to the conventional method that obtains to stretch.The barrier series of classification, it also produces shearing force for being placed into the pillar in the structure on the molecule that passes through, not only guarantee the initial stretching, extension of polymkeric substance by means of described barrier, and keeps stretching after described polymkeric substance passes barrier.
5.3 be used for the structure of stretching polymers
The structure (" elongated structure ") that is used to stretch DNA of the present invention comprises two parts: a conveyor zones and a polymkeric substance elongated region.Described conveyor zones is the passage of a broad, its introducing and draw described polymkeric substance elongated region.Described elongation zone comprises at least one of four major parts: (1) funnel; (2) has the structure of branched bottom; (3) has the passage of bending or curve; (4) limit the barrier in little gap, wherein said barrier is pillar or step especially.The present invention includes the combination of the variant of described four major parts and described major part itself.The combination of two or more described major part characteristics can produce additional design, and it is applicable in a kind of controllable mode and elongates and stretching polymers, especially DNA.In addition, several identical designs can parallel connection or series system repetition.
The example of structure (Fig. 1) that belongs to the scope of the invention includes but are not limited to:
I) has the funnel that aspect fluid velocity, non-linearly increases;
Ii) has the linear funnel that increases aspect fluid velocity;
Iii) have the funnel of the barrier in the little gap of qualification as the DNA elongated region;
Iv) has the funnel that aspect fluid velocity, non-linearly increases and limit the barrier in little gap;
V) has the funnel that aspect fluid velocity, increases and limit the barrier in little gap linearly;
The barrier size and the gap that vi) have mixing comprise the funnel of the gradient in barrier size and gap;
Vii) has the branched structure that increases the zone of fluid velocity from convergent passage;
Viii) has the branched structure that increases many zones of fluid velocity from many convergent passage;
Ix) has the branched structure of the barrier that limits little gap;
X) has at least one branched structure as the funnel of one of branch;
Xi) have the barrier size and the gap of mixing, comprise the branched structure of the gradient in barrier size and gap;
Xii) have the barrier in the little gap of qualification and the structure of bending or curve;
Xiii) has the structure that limits little barrier with periodicity (sinusoidal style, square wave repeat, transfer in a zigzag) gap;
Xiv) has the structure of the non-quadrilateral barrier that limits little gap;
Xv) have the structure of the barrier mixture that limits little gap, for example limit little gap and arrive sinusoidal style zone side by side, or one group of grid of triangle, annular or star region;
Xvi) has the structure that the barrier that limits little gap and funnel, branched structure or bending or curve combine;
Xvii) structure that has the bending in funnel shaped or turn;
What xviii) have bending or a curve limits the structure in little gap with barrier;
Xix) has the structure of the DNA elongated region of series connection;
Xx) has the structure of DNA elongated region in parallel;
Xxi) have many structures with transfer passage of elongation zone separately;
Xxii) have the structure that three-dimensional geometry is learned, it comprises the embodiment of other kinds; With
Xxiii) be the structure that comprises the closed circuit of DNA extension region.
Belong to further example explanation in Fig. 2 (a-1) of the structure of the scope of the invention.These comprise, comprise several embodiments of the stretched out structure of funnel, barrier, branch and cascaded structure; Two funnels with series connection of pillar; The embodiment of several compound pillars arrangements and branched structure; Asymmetric branched structure; Structure with the little barrier combination that limits little gap; Structure with polygon, rod and the combination of pillar barrier; The asymmetric bending structure; Branched structure with pillar; Big funnel structure with pillar; Funnel structure with pillar; Has the linear funnel structure that has and do not have pillar that increases aspect flow velocity.Fig. 2 (m) is the summary of some possible funnel structures.Usually, elongated structure of the present invention can have the length of 1 μ m to 2cm, preferred 1 μ m to 1mm, the height of 2 μ m to the width of 1mm and 0.1 μ m to 10 μ m.
Each of four major parts of following representation function polymkeric substance elongation and stretched out structure.
Funnel structure.Funnel structure is a tapered channel, and it applies shearing force with rule and continuous mode during along channel flow at polymkeric substance.Specific shearing force is by the type-restriction of channel architecture and shape.In one embodiment of the invention, passage is tapered channel (Fig. 3), and it originates in given width and is reduced to second width continuously, produces the shearing force that increases in the funnel part of described passage, and it is defined as:
du/dx＝(-Q/H)(dW/dx)(1/W
2) (13)
In one embodiment of the invention, described width linearity ground reduces, so dW/dx is a constant; In this embodiment, described shearing, du/dx increases when W reduces.In this embodiment, the angle of described funnel, when when the extension of stalk is calculated, preferably 1-75 °, at low viscosity solution DNA in TE (10mM TRIS, the 1mM EDTA) damping fluid for example, most preferably value is 26.6 °, the pH of described solution is 8.0.For linear funnel embodiment, the beginning width is preferably 1 micron to 1cm, and aftermost breadth is preferably 1nm to 1mm, depends on the polymkeric substance of being studied; For DNA, most preferably value is 50 microns and 5 microns respectively.
Described passage can also be arranged so that when fluid during by described passage described width reduce (Fig. 4) to advance the speed, the result shears when passing passage and increases.Like this decrescence provide especially good prevention polymkeric substance lax naturally protection because when the time pass and described molecule when described passage moves, the reacting force that they are increased stops their coiling again to be inclined to.In addition, the power gradient of described increase makes some design have dirigibility; Any polymkeric substance, it will run into enough big shearing force stretches this polymkeric substance and can not run into enough big shearing force described polymkeric substance is ruptured in described pyramidal structure in described pyramidal structure, can successfully pass described pyramidal structure and stretch.Needn't seek ideal or limiting force, only need an effective range for described polymkeric substance.In the embodiment that comprises pressure-drive fluid mobile (referring to following driving force), the shearing force that increases also provides for the speed to the maximum of constant pressure drop to be increased, because final speed is the function of cross-sectional area, and pressure drop is the function of cross-sectional area and passage length.Same little cross-sectional area (therefore big speed) can arrive (so pressure drop is less) than short distance.In a preferred embodiment, the width W of funnel is with 1/ (ax
n+ b) reduce, wherein n is that any a is the real number of non-zero greater than 1 real number, b is that real number and x are the distances (and being the direction of polymer flow) along described funnel length.The potential equation of taper of the shearing force funnel that increases comprises W=1/x
2, W=1/x
3, or the like.
In another embodiment, passage is designed such that shear rate is a constant, obtains a tapered channel, for example as shown in Figure 5.Acquisition is enough to fully to stretch the power of described polymkeric substance in the stroke of crossing channel constant shear rate will change (with reference to equation (12)) according to the length of passage.Therefore, for one very long for example＞passage of 1cm in stretching polymers fully, reasonably shear rate can be 0.01/s, but should value in the passage of very short for example＜10 a μ m stretching polymers hardly.Passage length can change significantly, preferred value be 10 μ m to 1cm and most preferably value be 1-2mm.In one embodiment, described passage is that 1mm length and shear rate are 0.075/s.
The shear rate of funnel can be determined by means of the distance of measuring between two known points of DNA chain.For example, the concatermer of λ DNA (concatamers) is used as the standard model of measuring shearing force.Unique sequence on each concatermer is used the hybridization probes mark with fluorescence mode.Therefore probe spacing on concatermer is from being the length of single λ dna molecular (48 kilobase).Physical distance between described probe uses image microscopy or flight time measurement to determine.For the λ DNA in natural solution, described physical distance is 14.1 μ m.This value is mated with the physical distance of actual measurement.For example, if measuring distance is 15.0 μ m, then shear rate can be calculated by the span that DNA experiences in described stretched out structure.The shearing force on DNA of prediction is when the dimension measurement of speed by DNA and passage (referring to equation 10), consistent with elongation and its unintentional nonlinearity rigidity of DNA.
The passage of branch.Second aspect of the present invention that is used to stretch and extends polymkeric substance is to set up branched structure, and it causes the variation of fluid flow rate or the variation of polymkeric substance directivity (vide infra, have the structure of bending or curve).Lateral access makes more fluid be fed to the main channel, causes the variation of fluid velocity and therefore causes polymkeric substance to stretch.The typical arrangement of branched bottom is shown in Fig. 6.The preferred total cross-sectional area of lateral access is equivalent to about 1% to 500% of described main channel cross-sectional area.Most preferably, total cross-sectional area of lateral access is about 50% of a described main channel cross-sectional area.In one embodiment, described lateral access exists with the style that repeats, and it causes entering each independent porch shearing force dilution of described main channel, therefore causes the approaching approximate value of constant-shearing force situation.This scheme has been given prominence to the merits and demerits of described lateral access.A shortcoming of this polymkeric substance elongate component is near the meet of described main channel and described lateral access, all to act on being pressed into of described main channel fluid and be dissipated in the relatively little zone.Therefore, this structure can not cause constant-Li situation.Yet, the advantage of this polymkeric substance elongate component is, because described additional fluid moves on same direction with the same in described main channel in described lateral access, therefore described power is not pure shearing force, but has suitable elongation components of flow.Pure shear, it is applied on the polymkeric substance by means of conical hopper, is the superposition of tensile force and revolving force, shown in Fig. 7 (a).Described tensile force on polymkeric substance quickens its motion on described fluid flow direction, makes that the part of the polymkeric substance be arranged in described elongation flow region is faster than the componental movement that still is positioned at the zone of stagnating more, thereby stretches described polymkeric substance.Described revolving force rotates or " rolling " described polymkeric substance on form, it can cause new winding of laying equal stress on certainly that partially overlap in of polymkeric substance stretching, extension on one's body.In the embodiment with stronger tensile force, the lateral access that for example is shown in 7 (b) connects configuration, and polymkeric substance tends to quicken to break away from from described meet, and it causes lower revolving force, therefore can better stretch.
Just as the skilled person will appreciate, described channel size can change and flow velocity can increase in the same area of micromodule.In fact, the method that flow velocity significantly increases before constant-cutting out section can not only stretching polymers, and guides its wall away from described passage.A configuration that comprises this embodiment of the present invention is shown in Fig. 8.In another embodiment, only introduce additional flowing from a side of described main channel, the polymkeric substance that will advance along described main channel is located to a side whereby.This positioning design can be used for guaranteeing that polymkeric substance is arranged by narrow detecting device in a wide passage.
Structure with bending or curve.The 3rd aspect of the present invention utilizes tortuous the acquisition to stretch.Run on its route when changing when fluid is flowing in, from the little right-angle array that bends to, the fluid in the described curve or the outside, turning will than fluid cost longer time of described curve or inboard, turning to walk around described turning (Fig. 9 (a)).This usually said " runway effect " can help stretching polymers.Such bending does not comprise ' T ' meeting point.At the rectangular section of a passage, polymkeric substance can stride across the fluid flow path that surpasses and flow, because described fluid advances with identical speed on each route, so it keeps configuration.In contrast, the distance of advancing when each fluid flow path is when a bending or corner depart from, and described polymkeric substance is stretched partly by means of velocity contrast.In addition, polymkeric substance tends to the stream line motion to higher-speed, even therefore described channel plot is got back to its initial direction, described polymkeric substance can fully not twine again, because it is in identical streamline partly.A kind of possible sequence of this stretching, extension is shown in Fig. 9 (b).Though this effect is not enough to stretch the molecule of overall length in single turning, it can little by little stretch specific zone, and the bending channel that fully repeats can stretch overall molecule.
A kind of tortuous more specifically mode is such embodiment, wherein the sinusoidal wave form (Figure 10) that is configured as of passage.In another embodiment, described passage is taked the form (Figure 11) of shape in a zigzag, perhaps, in embodiment further, take even the form that only has right angle corner (Figure 12) of " snake " shape, remain in a standstill and other unwelcome fluid dynamics problems although this sharp-pointed turning is tended to cause.For those embodiments, wherein said passage has flexuose turnover shape, and each bending preferably has 5-75 ° angle; For DNA, the preferred value at the angle that each is such is 26.6 (are 53.4 ° of angles effectively in flexuose turning point).Flexuose shape like this can be a round-robin, and wherein Wan Qu angle is always identical, perhaps can comprise different crooked styles.For flexuose turnover, described circulation can from little to 2 μ m to 1cm, be 20-50 μ m (1000 times to persistence length) for the DNA preferred value.Have the embodiment of sinusoidal wave form for those wherein said passages, amplitude to period ratio is 0.01-5 preferably.For any of these style, the number in cycle can be 1-500, and preferred value is 10.
In further embodiment, bending channel is used for producing the possibility of multiple detection.Work as detecting device, for example be arranged in the photomultiplier of the position-dependence in 1 * 256 array, when being provided with by the flow direction along described passage, described bending channel can be arranged like this so that it repeatedly passes detection zone at the position that limits.The polymkeric substance that is stretched is observed at several positions, produces in described system and repeats and error-checking.Such configuration is shown in Figure 13, and wherein fluid advances along passage 111, and at six positions, 112-117 is by detection zone 110.
Limit the barrier in little gap.The 4th aspect that helps to cause the structure of stretching, extension is barrier region.More generally describe as above-mentioned, barrier is by means of the cross-sectional area that reduces available described passage (producing local train on molecule) and serve as the intransitable physical barrier of big polymer coil, induces stretching, extension.The pillar configuration that example is an in fact stretching polymers is shown in Figure 14.
Described barrier can change aspect shape of cross section and the cross-sectional area.Term about barrier used herein " shape of cross section " and " cross-sectional area " unless otherwise stated, refer to the shape of X-Y projection of described barrier and the area of X-Y plane respectively, as shown in figure 15.In specific embodiment, described barrier comprises square pillar, cylinder, oval pillar or has the pillar of the rectangular cross section of any aspect ratio (comprising extremely long " grid "); In another embodiment, described barrier comprises the pillar that has as the polygonal shape of cross section of non-quadrilateral rule or irregular.In a preferred embodiment, described shape of cross section is leg-of-mutton.In another preferred embodiment, these shapes are changed to have concave surface (for example shallow U-shape) on the edge of the next direction of facing fluid.In another embodiment, having one of them size, to be longer than the preferred aspect ratio of pillar of the shape of cross section of another size be 2-20, more preferably 2-5.
In the middle of these barriers each can be placed to and flow to into any angle.In preferred embodiments, described barrier is aligned to or has plane perpendicular to described flow direction, with flow into 45, if require the preferred orientation of described polymer molecule, then use physically directs polymer towards other angles of destination direction.Preferably, one of them size is placed with the size of the length that makes them perpendicular to described flow direction than another long barrier.Another factor in described barrier layout is the grid that they are placed.If be placed on the square formation of repetition (Figure 16 (a)), some fluid flow path is influenced by described barrier hardly, and the polymkeric substance that does not stretch or stretch difference so perhaps can these streamline of approach and broken the barriers the zone and do not stretched.For preventing this point, next barrier (Figure 16 (b)) is placed in the placement that preferably misplaces of each continuous row in the gap of previous column, forces whole streamlines to have bending and causes stretching the molecule that all passes through.Described dislocation also can be less than described repetitive whole 50% so that each other row are as shown in the drawing not in identical arrangement; The for example per the 4th or the 6th row can have same arrangement, perhaps can not have the arrangement of repetition, as long as streamline is forced to crooked cut-through thing at some point.
The arrangement in flow process, have two other parameters relevant with barrier: the size of the passage between them, total Y-Z cross-sectional area of pillar is with respect to the Y-Z cross-sectional area (Figure 15) of passage, and wherein the both influences preferred barrier size.Channel width between barrier should be less than the diameter of the polymkeric substance that stretches, and preferably is not less than about 50 times of polymkeric substance diameter of stretching, extension, and increasing the probability of polymkeric substance by this passage, and inadhesion is at barrier region.The insufficient example that causes polymkeric substance not break the barriers of channel width is shown in Figure 17.On the other hand, this passage is preferably the same not wide with the diameter of the polymkeric substance of reeling, and a ball of string may break the barriers the zone and must not stretch under these circumstances.Therefore, the preferred interval height of barrier depends on analyzed polymkeric substance.Under the situation with length dna that 2nm chain diameter and winding diameter upwards change from about 1 μ m, preferably between 100nm and 800nm, most preferably value equals 500nm to channel width.For having the very polymkeric substance of minor diameter, can use gel to replace barrier region, its aperture size (channel width that is equivalent to described zone) arrives 1000nm for 1nm.
The total Y-Z cross-sectional area that is occupied by barrier the most directly influences the velocity gradient that appears between the barrier, and it impels stretching, extension.Therefore, preferably have bigger barrier Y-Z cross-sectional area and overall channel Y-Z cross-sectional area ratio (also claim packing factor, when representing with percentage, its ratio for the total area of the total area of pillar and passage takes advantage of 100) so that the velocity gradient maximization.On the other hand, attempt to enter a passage simultaneously, force too many material may cause blocking by relatively little gap if surpass a polymkeric substance.Therefore, be the consideration of these contradictions of balance, packing factor is preferably between 33% and 95%.This is the ratio that the area that blocks in a special modality and the total area are expressed as percentage.For example, has 1 μ m
2The pillar of Y-Z cross-sectional area has 3 μ m
2In the passage of Y-Z cross-sectional area, have 33% packing ratio, and 20 μ m
2Pillar is at 21 μ m
2Packing factor in the passage is 95%.For the most preferred packing factor of DNA between 50% and 80%.The example that too big barrier causes blocking is shown in Figure 18.
In order to alleviate the problem of the passage that polymer blocking is little in described pillar zone, different channel widths is used for certain embodiments of the present invention.In certain embodiments, finish by the size that changes barrier.In other embodiment, finish by changing packing factor.In other embodiment, change barrier size and packing factor.In such embodiments, polymkeric substance at first runs into passage between the wide barrier, runs into the passage (Figure 19) of reduced width subsequently, force they little by little become more extend to pass through smaller channels.In a preferred embodiment, channel width on flow direction in turn from about every passage 5 μ m to about every passage 1 μ m.In another embodiment, the pillar size is about 10 μ m from cross-sectional area on flow direction in turn
2To about 1 μ m
2In other embodiment, described barrier cross-sectional area can be changed into respectively with channel width and obtain similar effect, be that described barrier size can change with described channel size and can keep constant, perhaps described channel size can change with described barrier size can keep constant.In a preferred embodiment, all barrier has identical cross-sectional area, but packing factor increases on described flow direction.The cross-sectional area of pillar can be from 0.1 μ m
2To 1mm
2Change, preferably from 0.1 μ m
2To 10 μ m
2, more preferably from 1 μ m
2To 100 μ m
2, even more preferably from 1 μ m
2To 25 μ m
2Change; It depends on the size of the passage of the size of the polymkeric substance that is stretched and use.Such polymkeric substance pre-aligned is used for the possibility that reduces to tangle, and therefore more predictable stretching, extension is provided.
Barrier can also be manufactured into the structure into height or z-dimension, promptly introduces " step " to reduce described height by top and/or bottom at described passage.Replace the barrier crossing channel to place, as discussed above, described overall passage can change aspect height, when barrier when described width of channel is placed, the barrier of identical type is provided and centers on the shearing force of described barrier.In addition, aspect height, change and to finish more cheaply, because it is easier than managing to produce characteristic dimension at submicron-scale usually to use photoetching process to control etched height at submicron-scale.Be not subjected to the restriction of any theory, produced the effect identical in fact an ad-hoc location marked change in height, perhaps identical effect with the funnel of unlimited short length x with the single row of pillar.For so that the mode of the little package technique manufacturing of use standard is simulated funnel, described height change can be designed to several steps of existing along the length of described passage, rather than at the step at single position.In a preferred embodiment, single-step configuration reduces height to five/one of described passage.In other embodiment, the configuration with at least one step reduces described channel height from about 1/2 to about 1/100.In other embodiment, step is changed to the about 0.9 μ m of about 0.1-aspect height.
The combination of parts.In the further embodiment of the present invention, three general aspects of described structure, the combined use of shear-induced (that is, taper and branched bottom), complications and barrier-fill.For example, described constant-not only this is good in the stretching, extension aspect to shear tapered channel, and is good in the stretching, extension of the polymkeric substance that stretches in the zone that keeps having broken the barriers.Passage with tortuous profile can also dwindle on width to utilize both effects according to constant-shear pattern.In preferred embodiments, classification barrier region or arrangement architecture are used for stretching in advance described polymkeric substance, be accurate barrier part then, winding form or high-shear region, stretch up to arriving check point with maintenance with the cutting out section of finishing a stretching, extension and a constant-shearing or increase.
The applicant finds that especially effectively structure is the combination of the barrier region of tapered channel upstream.Described barrier region is used for stretching the DNA of random coil configuration, preferentially an end of molecule is delivered to the structure in downstream.Advantageously described barrier region is in the wide zone of passage, and is lower at this velocity ratio, makes the resistance put on folding molecule that center on or that kept by one of described barrier on the contrary be not enough to destroy described molecule.When the described barrier region of molecule wind through, an end will tend to guide described molecule and at first enter tapered channel.So described molecule will stretch further by means of the shearing force that flows by described tapered channel.Be not subjected to any theory constraint, the applicant finds, is subjected to the described part stretching, extension of barrier region influence and the stretching, extension in the terminal preposition tapered channel that coexists, and is especially effective in finishing the DNA stretching, extension.Relatively show, obtained stretching, extension preferably (referring to embodiment 2 and accompanying drawing 29 (a) and (b)) under similar flow and temperature conditions by described pillar zone and tapered channel are made up from the test figure of tapered channel and data from independent tapered channel with pillar zone, upstream.Described test figure shows, though tapered channel can stretch DNA, tapered channel is provided bigger significantly average stretching, extension and stretches the more DNA of vast scale with the structure that the pillar zone combines.
In preferred embodiments, barrier region, step or arrangement architecture are used to stretch in advance and arrange described polymkeric substance, are constant then or the shearing of increase or the part of elongation, to finish and to keep described stretching, extension up to arriving surveyed area.Preferably, described barrier region and tapered channel coupling are with flow (that is, speed reduces) that avoids shrinking.Therefore preferred pillar or step are positioned at or end at the gradual change part of described passage.
In a more preferred embodiment, described passage is a kind of structure of two funnels, and promptly it has the zone of the different tapering of having of two series connection.The example of two funnel structures is shown in Figure 20.In one embodiment, described two funnel structures further are included in a pillar zone in first conical region.In described two funnel configurations, (the rightest passage area among Figure 20) finished in extending in second conical region of polymkeric substance.It is preferred driving force that pressure-driven flows, because it is simply used with being easy to.
In a most preferred embodiment, described structure has a first passage zone, and it has the width of constant about 10 μ m and the height of about 1 μ m, wherein placed barrier region along described flow direction, with second channel zone of introducing, it is a funnel, with 1/x
2Decrescence, width from about 10 μ m to about 1 μ m and its height reduce in a single step at the inlet of described funnel, from about 1 μ m to about 0.25 μ m (Figure 21).The ratio of initial channel width and final channel width be preferably more than 10 and funnel part length preferably less than 1/2nd of original width.Described barrier region preferably includes 12-15 row's pillar at least, and it has the xsect that is substantially equal to 1 μ m, the wherein said packing factor that has increase on the described flow direction that comes.In one embodiment, six rows have from 0% to 50% packing factor that increases on flow direction, 12-15 row subsequently has 50% constant packing factor, and the row's that wherein said 12-15 row's subsequently center is contiguous distance is about 2 μ m (Figure 21).In another embodiment, described coming has the packing factor that increases continuously, from 0% to 80% on the flow direction.
5.4 be used for structure by means of the length selective polymer
Described as above-mentioned part, the pillar zone can be used for producing nonrandom polymkeric substance arrangement and effectively from an end of random coil isolating polymer chain, described random coil is the balanced structure of described polymkeric substance in solution.If it is the L place that a pillar zone is placed on apart from the tapered channel mouth, it can be to wish to keep or produce the Any shape that stretches, and for example straight, constant shearing or high-order moment, the structure that obtains can also be used to select molecule by means of length.This method is for example in Figure 22.
Figure 22 explanation is according to the sketch (referring to the method for manufacturing structure) in the pillar zone of method construct as described below, and it is positioned at shearing or extends before the funnel area that flows.Because described pillar loads the part of described passage,,, experience speed is reduced when described pillar regional movement enters the straight portion of described passage by the fluid of described passage motion.This speed reduces to produce contraction and flows, and promptly described polymkeric substance will be reeled in the zone that fluid velocity reduces again.Advancing and hook the dna molecular of pillar along described passage will be by described mobile stretching, extension.If the length of described molecule is equal to, or greater than the distance L of the starting point from described pillar to described conical region, it will discharge into described elongational flow zone from described pillar zone, in fact pass the zone that described fluid velocity reduces, and do not twine again, and will keep stretching, as in Figure 22, passing through the dna molecular illustrative.If described molecular proportion L is short, the dna molecular in Figure 22 2 for example, then it will leave described pillar, and still in the contraction flow region of described passage, and will promptly shrink this its becomes balanced line group.Therefore, length will be stretched more than or equal to the molecule of L, and length can not stretched less than the molecule of L.If detecting device is positioned in the outlet of described funnel, as shown in figure 22, the signal of the molecule (length is more than or equal to L) of coiled molecule (length is less than L) and stretching, extension will be differentiable.If for example described detector monitors intercalator-painted DNA, the molecule of contraction will produce short, strong pulse, and the molecule that fully stretches will produce long, not too strong signal.Therefore can make such structure, it can be separated into the population mixture of polymkeric substance two cohorts, being length is equal to, or greater than those of L less than those and the length of L, by simply with L, distance from the terminal in pillar zone to the conical region mouth, be set at a kind of like this length, promptly the length with the molecule that will detect its signal is identical basically.
In another embodiment, may wish given stretch in overall and uniformly detect signal from the molecule of all length at one.This can be undertaken by eliminating the contraction flow region, for example by means of the pillar zone admission passage that extends Figure 22, as shown in figure 23.Because described detecting device is positioned at feeder connection (as Figure 22), finish in this pillar zone, all molecules will be stretched by detecting device the time, therefore from the signal of whole molecules, no matter their length, with detected.In these embodiments, flowing keeps constant, because the area of the passage that area between described pillar and described pillar zone extend to is complementary.
5.5 design considerations
Stretch the structure type of considering and using.Different structures produces dissimilar DNA and stretches and extend.Exist and fasten (tethered) stretching, extension and evenly stretching, extension.Fasten stretching, extension and require to produce uneven power distribution, to produce elongation completely at flow profile at an end of molecule.Fasten the barrier and the directly generation of stretching the little gap of use qualification.On the other hand, evenly stretch more complicated and relate to the simulation widely of polymkeric substance dynamic perfromance.Evenly stretch on each unit that is defined in dna molecular and produce uniform tension.Be designed to produce the structure of even stretching, extension, be included in those that have constant shearing force on the x-direction of principal axis of described design, for example on flow velocity, have the funnel of non-linear increase.
Polymer sizes is considered.Structural design makes that they scalable (scalable) and some are general.Structure can increase and change relative size aspect size, to adapt to the polymer molecule of different length.Valuablely be of a size of several kilobase to the DNA of megabasse at least, though there is not the upper limit in the polymer molecule length that can be adapted to.The DNA of one megabasse has the length greater than 300 microns.Channel size can be made several millimeters.In such a way, complete chromosome (being of a size of the 50-250 megabasse) can processed and stretching, extension.
The structure of the passage on overall micromodule.The transfer passage that leads to the DNA elongated region can comprise such transfer passage, and it is in parallel, actinoid, branch, interconnection and loop closure.Transfer passage in preferred embodiments is wide passage, i.e. 1-1000 micron, and it leads to DNA and stretches and elongated region.
The method of manufacturing structure.The method for optimizing of making described project organization is, lithography, E-beam lithography for example, deeply-the uv lithography, photoetching process, (prefix of German words " Lithographie ", " Galvanoformung " and " Abformung " refers to lithography to LIGA, electroplate and molding) and the molding of resilient material.Made 2 and 3 dimensional organization by means of these technology.The other method that produces three-dimensional qualification passage comprises track-etching and molding technology.
Other the method that produces the nano-scale barrier comprises and relates to for example method of the cross-linked network of the self assembly of optics deposition, the localization polymkeric substance of colloid and polymkeric substance of chemical method.For example, the non-linear funnel that has the Ago-Gel of local deposits in funnel can produce the environment that control is stretched.
Conveying device.The structure that is used for stretching polymers is not unique useful parts that place passage.Being designed to advantageously to locate polymkeric substance is useful in a part of passage rather than the structure of another part, can guarantee that described polymkeric substance is supplied to specific stretched out structure or specific detection zone.Add the fluid (referring to branched bottom) to single passage except that aforesaid, can also be by means of forcing streamline mutually near finishing.The polymkeric substance that flow to be driven by fluid (for example pressure reduction and gravity are induced by means of any method of quoting as proof subsequently) will mainly (be used for the electrophoresis of electrically charged XC polymer along described fluid flow line, described polymkeric substance is along electric field line, and it can similarly be changed) motion.Irregular movement can cause that the part of chain is to contiguous stream line motion.Shrink or barrier if described streamline runs into, then described streamline becomes approaching more mutually around barrier, causes bigger chance to make the irregular movement of same side cause the variation of streamline.When described streamline (if described routing restoration to its initial width) when the opposite side of described structure returns to their primary leadings, the velocity gradient between described streamline is tended to polymkeric substance is dragged to streamline faster.Like this, the polymkeric substance of former stochastic distribution can be transformed into more well-regulated.For example, in one embodiment, big triangle in passage central authorities, the one side perpendicular to surface channel downstream, tend to make the direction location of polymkeric substance towards the center, this is because the polymkeric substance of original close wall laterally moves in described leg-of-mutton downstream by means of fluid and tends to the center of pulling to.In other embodiment, other shape is used to promote the location, for example criss-cross barrier, and wedge, and barrier region with dislocation, it helps the bigger passage of particular side guiding at passage.Should have position effect though have the passage of simple bending, this looks like intuitively, yet included velocity gradient is in fact quite little, and its independent effect is quite little.
In structure, improve the method that stretches.In other embodiments of the present invention, scissors-induce the validity of mode to be improved by means of the viscosity that increases solution.The power of the reality that produces by means of the contraction of passage is directly proportional with solution viscosity.In certain embodiments, solution viscosity increases by adding one or more viscosity modified components.Glycerine (at room temperature viscosity nearly 900cP), if its not with described polymkeric substance chemically reactive, can add aqueous solution with concentration up to 70% (w/v).Also can add sugar, for example sucrose, wood sugar and D-sorbite.Can also add water-soluble polymers, for example polyglycol.Under the DNA situation, high molecular weight polyacrylamide, polyethylene oxide or long-chain glycan (even in the concentration that is low to moderate 0.01% (weight)) can increase viscosity in aqueous solution and not change the structure of the DNA that will characterize.
Described viscosity also can change by adding the polymkeric substance that an amount of quilt characterizes, but it can not detected by the detection zone of described structure.For example, if FRET carries out on the dna molecular of external ground mark, the dna molecular of then additional is not external ground mark can be added to the polymer solution of described mark to increase viscosity.Like this, only detected the and unmarked DNA of labeled molecule only changes solution viscosity, signal that can the overslaugh labeled molecule.
In another embodiment, viscosity increases by reducing temperature; For example the viscosity of pure water is bordering on and doubles when near freezing point.Except that increasing viscosity, the reduction temperature is used to make Brownian movement to minimize and prolongs slack time.Work as aqueous buffer solutions, 1X TE solution (10mM TRIS, 1mM EDTA) for example, during from variation of ambient temperature to 4 ℃, stretching has suitable improvement.
Driving force.Be used to make described polymer motion can comprise physics, electricity, heating power or chemical force from any equipment by the driving force of described structure.The simplest driving force is flowed by capillary-driven, when sample solution contacts at first with described device.Though the surface energy that comprises can provide in described passage at a high speed, the control of flow is limited in this mode.
Utilize chemical potential can obtain indirect therefore limited control.An advantage setting up concentration gradient provides extremely slowly, stable flow velocity.This is by producing the excessive in a large number of material and carrying out, control based on excessive concentration causing fluid to flow through described structure consumes described diffusion after opposite side material at it in a side of described structure.Described polymkeric substance flows through described structure with the fluid of inducing by means of described migration material.
An embodiment preferred is directly controlled flowing of fluid.In such embodiment, the entrance side formation pressure head in structure impels the direction of flow distally, and the latter opens into atmospheric pressure or maintains the pressure of attenuating.Described pressure head can be from any device that utilizes physical force, for example syringe pump.At present, syringe pump distributes the scope until 100pL/s, and the flow that requires in device may be under 1pL/s, therefore " bypass " with big cross-sectional area may must be set up, increase the flow of matching requirements like this and can use commercially available device control, only lose the sample of some volumes.In another embodiment of control pressurer system, in the device of pressure drop less than atmospheric pressure, an end vacuum attraction of system little by little sucks the material by described construction stretch.Inducing with the mobile pressure drop that needs of the speed that requires is channel geometry (particularly Zui Xiao cross sectional dimensions) and function of speed, but for millimeters long, the flowing of 100 microns per seconds in the dark passage of micron (otherwise be very wide for most of devices), be typically about 10psi.In another embodiment, the pressure head of described passage first end and the combination of the vacuum of described passage second end be used to promote polymkeric substance from first end to second end.
In other embodiments, control the polymkeric substance that flows by described fluid by means of set up thermograde in every side of stretching area.Therefore natural convection is set up by the fluid of stretching area mobile.Because it is very difficult setting up and control thermograde at the micro-meter scale of these device operations, so this method,, be preferably used for very low fluid flow as the chemical potential method.
In another embodiment, the flowing of polymkeric substance for charged polymkeric substance DNA for example, controlled by means of setting up electric field, and it acts on the electric charge of polymkeric substance, and does not act on the fluid (if it is uncharged) that centers on.Described electric field preferably forms by the electrode of two opposite-chargings of existence in solution, but the electrode of total series can be used for setting up more complicated or uniform field pattern.Described polymkeric substance is along electric field line rather than stream line motion (in some cases, have incoherent variation, depend on the electric density of the physical layout and the described solution of described micromodule).This might damage stretching, extension, if the material that the solution that centers on comprises on the contrary-charges, its flow in the opposite direction (electric power flows), perhaps the surface charge on described conduit wall produces the ion flow (electric osmose flows) along described wall, and the both may induce at the fluid on the opposite direction flows and produce viscous force on described polymkeric substance.Yet in low conductivity solution, coated with avoiding surface charge, oppositely viscous force has inappreciable influence to the driving force of electrophoresis to wall by suitably, makes described polymkeric substance can advance by described structure and stretched.In addition, utilize suitably charged wall surface, electric osmose flows and can be reversed to provide viscous force, and it helps described electrophoresis to stretch.1000 to 2000V/m field intensity causes the polymer velocity of 100 microns per second scopes being suitable for.
Under electrophoresis and pressure-driven power situation, the device that produces described driving force physically separates with described stretching area usually.Electrode be set up at a distance of described stretching area several millimeters to many centimetres far, power supply then is positioned at even the farther place.Described syringe pump though advantageously approach described stretching area as far as possible to minimize the pressure drop that needs, tends to be placed on outside the described device, because its volume is big.In fact, for structure adaptability, preferably only described stretching, extension and detection architecture itself are placed on the little micromodule, preferably the length of side is not more than 2cm, perhaps little of the 1mm square, most preferred size (from the angle of operation) is taken advantage of 1cm for about 1.5cm, and thickness is 0.2cm.On this base material, various fluid flowing passages are set.In such micromodule, can on described base material, place 1-160 passage comfortably, 30-40 the balance that passage can be obtained: passage stop up or base material crackle situation under repetition and once only a passage be in the detection visual field (having the 60X object lens usually).
Base material.The condition that the selection of the base material that uses will be fit to solution and use in analysis includes, but are not limited to extreme salinity, acid or alkali concn, temperature, electric field and for the transparency of the wavelength that is used for optical excitation or emission.Described substrate material can comprise those relevant with semi-conductor industry, for example fused silica, quartz, silicon or gallium arsenide, perhaps for example polymethylmethacrylate, dimethione, teflon, polycarbonate or Polyvinylchloride of inert polymer.Because its transmission performance under various wavelength, quartz is an embodiment preferred.
Utilize and quartzyly to mean that as the base material that is used for aqueous solution the surface in contact with described solution has positive charge.When utilizing charged molecule work, particularly under deposition condition, wish to have neutral surface.In one embodiment, a coating is applied to described surface causes described electric charge with elimination interaction.Described coating can be purchased (capillary coating of Supelco, Bellafonte PA), perhaps by utilizing the silane that at one end has functional group to apply.Described silane is terminal will irreversibly can further to react the coating that requires to produce with glassy bond and described functional group effectively.For DNA, silane with polyoxyethylene can prevent interaction and the not further reaction between polymkeric substance and described wall effectively, can participate in polyreaction to produce the polyacrylamide coating with silane with acrylamide group, it does not only interact with DNA, and the electric osmose that is suppressed at during the electrophoresis flows.
Described passage can be by many technical construction on described base material, and many semi-conductor industries that derive from depend on selected base material.These technology include but are not limited to, and photoetching process, active-ion-etch, wet-chemical etch, electron beam are write, laser or air ablation, LIGA and injection molding.Various these technology that are applied to polymkeric substance-processing micromodule have been discussed in following document, comprise (Analytical Chemistry 1992 (64) 1926-1932) such as Harrison, Seiler etc. (Analytical Chemistry 1993 (65) 1481-1488), (AnalyticalChemistry 1995 (67) 2059-2063) such as Woolley etc. (Proceedings of the National Academy ofSciences 1994 November (91) 11348-11352) and Jacobsen.
Other consideration.In embodiment preferred of the present invention, in given channel plane height, be uniform basically at the rectangular channel medium velocity.This is correct during less than width when the highly significant of described passage ground, make non--sliding condition on described wall cause the parabolic velocity profile of viscosity-induce, this is significant in altitude axis, only stays the next little frontier district than slow flow in width axes.In length and breadth ((width/) than for about 10 or bigger be that such embodiment is needed, according to the approximate (Deen of lubrication theory, Analysis of Transport Phenomena, NewYork:Oxford UniversityPress, 1998.275-278).In addition, when using micro objective in optical system, little height helps to detect.Typical object lens can have 500nm and arrive several microns depth of focus, though so the height of passage can for 50nm to 100 μ m, as long as described aspect ratio is about 10, to adapt to described analyzed polymkeric substance, described embodiment preferred has the channel height of 200nm to 1 μ m, makes that the material that all passes through in passage will and be observed on focus exactly.
The present invention also comprises such embodiment, and wherein said passage is not the plane, and makes with the three-dimensional channel manufacturing technology.In such embodiments, constant scissors is not only induced from sidewall, and induces from the gradient channel height.Similarly, in further embodiment, the combination of structure has the power that acts on an axle and acts on another another power.In some such embodiments, barrier region is crossed over described width of channel, and its height reduces in conical in shape.In other embodiment, tortuous in single plane, interior to-helical design, it also reduces aspect channel width, be used to produce shearing force, it is at central feeding, enters the vertical outlet of described device by the hole in described material bottom, detects the inlet near described hole.When being in vertical direction when structure, gravity is used to some embodiment to help to produce velocity contrast in fluid.(it should be noted that independent gravity deficiency with stretching polymers or it is flowed, because the power that is added on the 100kD polymkeric substance just surpasses 10
-18N; Any effect of gravity will be experienced by viscous force by described molecule.)
6. embodiment
6.1 embodiment 1: be used to stretch DNA micromodule manufacturing and
It is being used for detecting from the application of the device of the DNA emitted fluorescence of mark
Experimental provision.The sensitive optical devices that are used to detect are shown in Figure 24.This device utilizes confocal fluorescent illumination and detection.Confocal illumination is illuminated little optics volume (about the millimicro microlitre).Using little probe volume minimizes Rayleigh and Raman scattering.Light beam from the 1mW Argon ion laser passes through laser line filtrator (514nm), is sent to dichronic mirror, by 100 * 1.2NA oil immersion objective, arrives sample then.Fluorescent mark on DNA can be comprise following several dyestuffs a kind of: Cy-3, tetramethylrhodamin, rhodamine 6G and Alexa 546.In addition, can use for example TOTO-3 (molecular probe) of intercalator dyestuff., focus on the 100 μ m pin holes by dichromatic, a narrow band logical (for example OmegaOptical) from the fluorescent emission of sample,, finally focus on (EG ﹠amp on the avalanche photodide of photon counting mode by aspheric lens; G Canada).Output signal multichannel scalar (EG ﹠amp; G) collect and use Pentium III Computer Analysis.Confocal device is suitable for comprising the quantitative Application of flight time.Such application is included in DNA and goes up measuring distance, certification mark order and determine degree of drawing in DNA.Can use this device and detect single fluorescence molecule.For the application that needs image, it is suitable using the device of the CCD (ICCD, Princeton Instruments) that is arranged on the reinforcement on the microscope.
The manufacturing of micromodule.On 0.090 inch thick quartz substrate, by photoetching process and beam methods, generated one group of constant-shearing passage, it has the design shear rate of 0.085/s, by 1.5 microns barriers guiding of 2 microns of two row's spacings.At first (5 parts of deionized waters are than 1 part of 30% ammonium hydroxide/30% hydrogen peroxide by being placed on the RCA solution that is heated to 80 ℃ for described base material, afterwards both are from Sigma Chemical Co., St.Louis purified in MO) in 20 minutes, flowed down drying at nitrogen then.Then will (MA) Xi Shi Shipley S1813 photoresist be spin-coated on quartz surfaces last 45 second under 3250rpm in spinner for Shipley, Newton, then 90 ℃ of curing 0.5 hour in baking oven with R type thinning agent with 2: 1 ratios.Then with coarse constant-shear pattern by being exposed to 12s under the mercury vapor lamp contact print on described surface, for example from Carl Zeiss, in the contact photoetching machine of Germany, then with 5: 1 ratios with 351 developers (Shipley) of deionized water dilution in rinsing 30s, further rinsing in deionized water flows down drying at nitrogen then.After the UV-of 10s ozone purification, described base material is carried out etching in 40 minutes with CHF3 in Reactve Ion Etch (RIE) machine.After another time washed in RCA solution, in spinner, under 2000rpm, polymethylmethacrylate (650MW) is diluted to 3% solution and is spin-coated on described surface last 45 second in chlorobenzene.Described coating in 180 ℃ of curing one hour, adds the chromium layer of 60 then in evaporator in baking oven.Carry out electron beam and write to make fine structure, for example barrier row carries out the chromium etching and uses rinsed with deionized water at the REI machine then.Then with described base material at the isopropyl alcohol that is heated to 21 ℃: 2 of methyl isobutyl ketone: immerse 90 seconds in the 1v/v solution,, carry out another time UV-ozone purification then to develop.In the REI machine, carry out another time CHF then
3Etching is succeeded by the RCA solution washing.
(Fisher Scientific, Pittsburgh is PA) with deionized water rinsing and dry under nitrogen will to be of a size of the cover glass of 45mm * 50mm * 0.15mm.With 10: (solution NY) on 4000rpm is spin-coated on described cover glass 60 seconds, solidified two hours in 80 ℃ in spinner the RTV615A:RTV 615B silicones of 1w/w then for General Electric, Schenectady.The silicones flat board that will have the hole of wherein settling micromodule places on the cover glass, and it is exposed in the blood plasma clearer of 30W 50 seconds then, to produce water-wetted surface.It is dull and stereotyped and with the rinsing in deionized water of described cover glass to remove described silicones then, dry under nitrogen then.The micromodule that will prepare fully is placed on the described cover glass carefully then.
Be used to monitor device to the pulse of picture-dependence from the DNA that stretches.As shown in figure 25, induction system is supplied with part 151 by polymkeric substance and is formed, it is driven by micromodule 152 (with reference to foregoing description) by syringe pump 150, wherein polymkeric substance stretches and is excited by the laser beam from laser instrument 154, it detects by photodetector 153 and analyzes with computing machine 155, and this computing machine is control pump 150 and detecting device 153 also.
The fluorescent emission of monitoring in the DNA that stretches.Escherichia coli T 4 phage DNA (Sigma, St.Louis, MO) by (base-pair: dyestuff) ratio added 4040-1 and carries out mark, cultivates one hour, then at 0.5X TBE electrophoretic buffer (45mM TRIS with 5: 1,32.3mM boric acid and 1.25mM EDTA, pH8.3, all from Sigma, St.Louis, MO) be diluted to 1/50th, 000 in.
Then a microlitre sample is drawn on cover glass with dropper, it is close to micromodule, is imported in the passage by means of capillarity this its.Described micromodule and cover glass are placed on fluorescent microscope (from the Microphot series of the Nikon) dressing table, and this fluorescent microscope is being equipped 60X plano apo lens (from for example Nikon or Carl Zeiss).Excite to come from mercury-arc lamp, have Nikon B2A light filter, to guarantee near the 490nm of YOYO-1 peak value excites, enough exciting being arranged.The emission that surpasses 520nm is captured by described B2A filter set and by the video camera (C2400-08 of Hammamatsu) or the ccd video camera of silicon-reinforcement.Image from video camera passes through the image capture card (for example from NationalInstruments, Austin, the PCI-1408 of TX) outputs in the computing machine, and analyze with image processing software, it is a written program customized, on screen, discern DNA, and calculating pixel is to determine polymkeric substance length according to brightness with respect to background.
Various dna moleculars (Figure 26) in this device, have been observed.The constant cutting out section of the micromodule in Figure 27 (a-g) has shown the dna molecular (63 microns) of the about 190kb that stretches.A DNA who fully stretches is shown in Figure 28 in described micromodule.This molecule is measured as 139 microns, perhaps 535kb.
Data.Will be with the small sample (half microlitre) of the painted T4 DNA (Sigma) of YOYO-1 (molecular probe), input has the rectangular funnel part together with pillar and the micromodule that moves under capillarity.Observe with 100WHg lamp excited sample and with SIT video camera (HammatsuC2400-08).Be sent to image capture card in Pentium level computing machine from the vision signal of video camera, move LabView software customized, it determines with the pixel to be the length of the DNA fragment of unit according to its speed and time of spending in study area.Be considered to fragment and automatically get rid of less than 30 microns length, it causes, and at sample about two minutes are in service only to obtain ten data points.Use the conversion of known amplification level, find that DNA is that 50.6 μ m are long, between 42 and 62 μ m.A histogram is shown in Figure 29.Described length is shorter than the desired value of the 71.1 μ m of painted 164kbp T4 DNA a little, illustrates to stretch in this design not finish fully.
6.2 embodiment 2: use the stretching, extension of the phage DNA of apparatus of the present invention
Used two kinds of different devices to obtain to be shown in data among Figure 30 (a) and 30 (b).The device that use is shown in Figure 20 obtains to be shown in the data of Figure 30 (b).The device that is used to obtain Figure 30 (a) data has and the identical channel boundary (that is, the size of two conical regions of two-funnel device is than identical) of device that is used to obtain Figure 30 (b) data, does not just have pillar in described structure.
Use aforesaid photoetching method, (Hoya Corp., San Jose CA) are etched into the style with Figure 20 with the fused silica sheet.Described use scribing machine (for example from DiscoCorp., Santa Clara CA) is cut into 1 centimetre of micromodule of taking advantage of 2 centimetres, then by means of heat bonding adhere to the fused silica cover glass (for example from Esco, Oak Ridge, NJ).
To have 48.5 kilobase homogeneous length (promptly, the expection stretched length be the 16-17 micron) double-stranded λ DNA (Promega, Madison WI) carries out mark by 3 μ MTOTO-3 iodide (molecular probe, the Eugene OR) intercalative dye that adds similar quantity, then at 1X TE damping fluid (10mM TRIS and 1mM EDTA, pH8.0, all from Sigma, St.Louis, MO) be diluted to about 1/50th, 000 in.For the two strands 48.5 kilobase dna samples that use here, be 21 μ m (approximately growing 30%) than uncoloured DNA with the expection spread length of the painted λ DNA of intercalative dye.
The microscope stage that micromodule and cover glass are placed fluorescent microscope (for example, Microphot series from Nikon), this fluorescent microscope is equipped with 100X plano apo lens (for example from Nikon, Carl Zeiss) and filter set, it (has for example carried out optimizing for being used for TOTO-3, from Omega Optical, Brattleboro, the XF-47 of VT).Excite to come from 633nm helium-neon laser (for example), focus on two points that are arranged in the microchannel on the identical streamline from Melles Griot.Make sample in the input of the porch of described passage by means of capillarity, and utilize vacuum at the described micromodule other end (produce by means of vacuum pump, Welch Vaccum for example, Skokie IL) continues described flowing.When dna molecular passed through laser spot, the emission that surpasses 650nm was accepted by described filter set and by a pair of confocal detecting device that is arranged on the described luminous point.Flight time between described detecting device is used to determine speed, and it used with the residence time in a laser spot, to calculate the length of molecule.
This result of experiment shows, two-the funnel device that comprises pillar makes the two strands of 48.5 kilobase, the λ DNA of dye coloring be stretched over the length of about 19.5 μ m (Figure 30 (b)), and the two-funnel device that does not have a pillar only makes DNA be stretched over the length (Figure 30 (a)) of about 10 μ m.Therefore, though there is not stretching, extension in DNA in having the tapered channel of pillar, on an average, DNA only is stretched over half just over its total length, and considerably less individual molecule fully stretches, as by histogram among Figure 30 (a) wide distribution proved.By contrast, in having pillar zone and downstream tapered passage structure combining, on an average, molecule is stretched over and approaches total length, and most of molecule is in 20% scope of their full extension length of expection.Therefore, two-funnel device with pillar can stretch DNA better than the same apparatus that does not have pillar.In addition, this device can be more equably than the two-funnel structure that does not have pillar and stretching polymers effectively.
7. the reference of quoting
Draw at the full text of these all references that will quote and to be reference, on whole meanings, its scope is equal to each independent publication or patent or patented claim and specifically and is respectively indicated it is drawn on whole meanings in full and be reference.
Under the situation that does not break away from the spirit and scope of the present invention, can carry out many improvement and change to it, this is conspicuous to those skilled in the art.Specific embodiment described here only is to provide for example, and the present invention is only limited by the coordinator of the four corner of claims and claim.
Claims (330)
1. integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises a tapered channel, described tapered channel reduces on width to second end linearly from first end, with wherein said at least a polymkeric substance, when existing, moving to the direction of described second end from described first end along described tapered channel;
Whereby when described at least a polymkeric substance in described fluid sample when described tapered channel moves, shearing force is applied on the described at least a polymkeric substance.
2. the integrating device of claim 1, wherein said tapered channel angle with 1-75 ° on width reduces, and described angle is defined in the angle of described first end with respect to the constant width passage.
3. the integrating device of claim 2, wherein said angle equal 26.6 ° and described at least a polymkeric substance and comprise DNA.
4. the integrating device of claim 1, the width of wherein said first end is that the width of 1 μ m-1cm and described second end is 1nm-1mm.
5. the integrating device of claim 4, the width of wherein said first end equals 50 μ m, and the width of described second end equals 5 μ m and described at least a polymer samples comprises DNA.
6. the integrating device of claim 1, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
7. the integrating device of claim 6, wherein said conveying equipment comprises transfer passage, described elongated structure is introduced and drawn to described transfer passage.
8. the integrating device of claim 1, it further comprises makes described at least a polymkeric substance, when existing, the equipment that in described elongated structure, moves.
9. the integrating device of claim 8, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
10. integrating device, it comprises:
A kind of elongated structure that comprises tapered channel, described tapered channel reduces on width to second end linearly from first end, described tapered channel comprises at least a polymkeric substance in fluid sample, described tapered channel is configured such that a shearing force is applied on the described at least a polymkeric substance when described at least a polymkeric substance during from described first end to described second end motion.
11. integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises a tapered channel, described tapered channel from first end to second end on width to reduce greater than linear velocity, with wherein said at least a polymkeric substance, when existing, moving to the direction of described second end from described first end along described tapered channel;
Whereby when described at least a polymkeric substance in described fluid sample when described tapered channel moves, shearing force is applied on the described at least a polymkeric substance.
12. the integrating device of claim 11, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
13. the integrating device of claim 12, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
14. the integrating device of claim 11, it further comprises the equipment that described at least a polymkeric substance in described fluid sample is moved in described elongated structure.
15. the integrating device of claim 14, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
16. an integrating device, it comprises:
A kind of elongated structure that comprises tapered channel, described tapered channel reduces with the speed greater than linearity on width to second end from first end, described tapered channel comprises at least a polymkeric substance in fluid sample, described tapered channel be configured such that when described at least a polymkeric substance when the direction from described first end to described second end is moved, shearing force is applied on the described at least a polymkeric substance.
17. integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises a tapered channel, described tapered channel reduces at width to second end from first end, with wherein said at least a polymkeric substance, when existing, moving to the direction of described second end from described first end along described tapered channel;
Whereby when described at least a polymkeric substance in described fluid sample when described tapered channel moves, shearing force is applied on the described at least a polymkeric substance, wherein said shearing force produces constant shear rate.
18. the integrating device of claim 17, the width of wherein said tapered channel satisfies equation: W=(SHx/Q+C)
-1, wherein W is described width, and S is described shear rate, and Q is that total flow and C is constant.
19. the integrating device of claim 18, wherein said shear rate is at 0.01s
-1-10s
-1Between.
20. the integrating device of claim 19, wherein said shear rate equals 0.075s
-1
21. the integrating device of claim 18, the length of wherein said tapered channel are 1mm-2mm.
22. the integrating device of claim 18, wherein said fluid sample are maintained at the following temperature of environment temperature.
23. the integrating device of claim 17, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
24. the integrating device of claim 23, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
25. the integrating device of claim 17, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
26. the integrating device of claim 25, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
27. an integrating device, it comprises:
A kind of elongated structure that comprises tapered channel, described tapered channel reduces at width to second end from first end, described tapered channel comprises at least a polymkeric substance in fluid sample, described tapered channel be configured such that when described at least a polymkeric substance when the direction from described first end to described second end is moved, shearing force is applied on the described at least a polymkeric substance, and wherein said shearing force produces constant shear rate.
28. integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, and wherein said elongated structure comprises that one is used to hold the centre gangway of fluid and is used to hold many lateral access that fluid is connected to described centre gangway; With
Wherein said at least a polymkeric substance when existing, moves on prolonging direction along described centre gangway.
29. the integrating device of claim 28, wherein said many lateral access are directed the fluid that makes from described many lateral access injections and move along described centre gangway on described prolonging direction basically.
30. the integrating device of claim 28, wherein said centre gangway have a central authorities-cross-sectional area and described many lateral access have total side-cross-sectional area, it is the 1%-500% of described central authorities-cross-sectional area.
31. the integrating device of claim 30, wherein said total side-cross-sectional area equals 50% of described central authorities-cross-sectional area.
32. the integrating device of claim 28, wherein said many lateral access are arranged in the mode that repeats.
33. the integrating device of claim 28, wherein said centre gangway is taper.
34. the integrating device of claim 28, wherein said fluid are maintained at the following temperature of environment temperature.
35. the integrating device of claim 28, at least one of wherein said many lateral access is taper.
36. the integrating device of claim 28, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
37. the integrating device of claim 36, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
38. the integrating device of claim 28, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
39. the integrating device of claim 38, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
40. an integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises:
(a) a kind of elongated structure;
(b) transfer passage of introducing and drawing described elongated structure, it carries described at least a polymer samples in described fluid to described elongated structure; With
(c) be used for making described at least a polymkeric substance at described fluid sample, when existing, the equipment that in described elongated structure, moves;
Wherein said elongated structure comprises that a centre gangway and many being used to that is used to hold fluid holds the lateral access that fluid is connected to described centre gangway; With
Wherein, when described at least a polymkeric substance existed, described equipment made described at least a polymkeric substance move on prolonging direction along described centre gangway.
41. an integrating device that is used for extending in the DNA of fluid sample, it comprises:
(a) a kind of elongated structure;
(b) be used for the described DNA that carries at described fluid sample equipment to described elongated structure; With
(c) be used for the described DNA that makes at described fluid sample, when existing, the equipment that in described elongated structure, moves;
Wherein said elongated structure comprises that a centre gangway and many being used to that is used to hold fluid holds the lateral access that fluid is connected to described centre gangway; With
Wherein, when described DNA existed, described equipment made described DNA move on prolonging direction along described centre gangway.
42. an integrating device, it comprises:
A kind of elongated structure, this elongated structure comprises a centre gangway that is used to hold fluid, and described centre gangway comprises that first end and second end and many being used to hold the lateral access that fluid is connected to described centre gangway, described centre gangway comprises at least a polymkeric substance in fluid sample
Wherein said at least a polymkeric substance is moving to the direction of described second end from described first end.
43. integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, and wherein said elongated structure comprises that one has at least one crooked passage and wherein said at least a polymkeric substance, when existing, move along described passage.
44. the integrating device of claim 43, wherein said passage has sinusoidal shape.
45. the integrating device of claim 44, amplitude that wherein said sinusoidal shape has and period ratio are 0.01-5.
46. the integrating device of claim 43, wherein said passage comprise many straight line portioies that connect with non-zero angle.
47. the integrating device of claim 46, wherein said many middle each of straight line portion have identical length and equate in size with described non-zero angle and alternately variation on symbol that therefore described passage has flexuose turnover shape.
48. the integrating device of claim 47, the size of each in the middle of the wherein said non-zero angle is 5-75 °.
49. equaling 26.6 ° and described at least a polymer samples, the size of each in the middle of the integrating device of claim 48, wherein said non-zero angle comprises DNA.
50. the integrating device of claim 43, wherein said passage comprises many right-angle bendings, makes described polymkeric substance move along the route of the repetition of two 90 ° of turnings and two-90 ° of turnings.
51. the integrating device of claim 43, wherein said passage comprise the round-robin shape and have the periodicity of 1-500.
52. the integrating device of claim 51, wherein said periodicity is 10.
53. the integrating device of claim 51, it further comprises the equipment that detects described at least a polymkeric substance along detection zone, and wherein said checkout equipment is located so that described passage repeatedly passes described detection zone in the position that limits.
54. the integrating device of claim 43, the equipment of the described at least a polymkeric substance of wherein said conveying comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
55. the integrating device of claim 43, wherein said passage holds a kind of solution, and described solution remains on the following temperature of environment temperature.
56. the integrating device of claim 43, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
57. the integrating device of claim 56, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
58. the integrating device of claim 43, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
59. the integrating device of claim 58, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
60. be used for extending in the integrating device of the DNA of fluid sample, it comprises:
(a) a kind of elongated structure; With
(b) be used for carrying the described equipment that arrives described elongated structure at the DNA of fluid sample;
Wherein said elongated structure comprise have at least one crooked passage and
Wherein said DNA when existing, moves along described passage.
61. the integrating device of claim 41, it further comprises makes described DNA, when existing, and the equipment that in described elongated structure, moves.
62. an integrating device, it comprises:
A kind of elongated structure, this elongated structure comprise having at least one crooked passage, and described passage comprises first end and second end, and described passage comprises that at least a polymkeric substance in fluid sample is used to stretch described at least a polymkeric substance,
Wherein said at least a polymkeric substance is moving to the direction of described second end from described first end.
63. integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises a tapered channel, along the described at least a polymkeric substance of this tapered channel, when existing, on flow direction, move and wherein said passage comprises the barrier of many described at least a polymer motion.
64. the integrating device of claim 63, wherein said tapered channel reduces on width to described second end linearly from described first end.
65. the integrating device of claim 63, wherein said tapered channel reduces with the speed greater than linearity on width to described second end from described first end.
66. the integrating device of claim 63, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
67. the integrating device of claim 66, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
68. the integrating device of claim 63, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
69. the integrating device of claim 68, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
70. integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises a centre gangway, along the described at least a polymkeric substance of this centre gangway, when existing, on flow direction, move and barrier that the many lateral access that is connected to described centre gangway and wherein said centre gangway further comprise many described at least a polymer motion.
71. the integrating device of claim 70, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
72. the integrating device of claim 71, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
73. the integrating device of claim 70, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
74. the integrating device of claim 73, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
75. integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises that has at least one crooked passage, along the described at least a polymkeric substance of this passage, when existing, on flow direction, move and wherein said passage comprises the barrier of many described at least a polymer motion.
76. the integrating device of claim 75, wherein said passage comprise the round-robin shape of the periodicity with 1-500.
77. the integrating device of claim 76, wherein said round-robin shape is a sinusoidal curve.
78. the integrating device of claim 76, wherein said round-robin shape is flexuose turnover.
79. the integrating device of claim 75, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
80. the integrating device of claim 79, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
81. the integrating device of claim 75, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
82. the integrating device of claim 81, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
83. integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises a passage, along the described at least a polymkeric substance of this passage, when existing, move on flow direction and wherein said passage comprises many pillars, at least one of described pillar has non-tetragonal polygonal cross sectional shape.
84. the integrating device of claim 83, at least one of wherein said many pillar comprises the edge of spill, the direction that the edge facing fluid of wherein said spill comes.
85. the integrating device of claim 83, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
86. the integrating device of claim 85, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
87. the integrating device of claim 83, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
88. the integrating device of claim 88, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
89. integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises a passage, along the described at least a polymkeric substance of this passage, when existing, on flow direction, move, comprise the barrier of many described at least a polymer motion with wherein said passage, described many barriers are with a series of registration, each described row locatees perpendicular to described flow direction, with each continuous row and last misarrangement position, whereby along described flow direction, the overlap extension in the gap that forms by two adjacent barrier in described last row of at least one part that is not equal to 1/2 the multiple of one of described barrier.
90. the integrating device of claim 89, wherein said passage is taper.
91. the integrating device of claim 89, it further comprises many lateral access that is connected to described passage.
92. the integrating device of claim 89, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
93. the integrating device of claim 92, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
94. the integrating device of claim 89, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
95. the integrating device of claim 94, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
96. an integrating device, it comprises:
A kind of elongated structure that comprises passage, described passage comprises:
(a) first end and second end;
(b) barrier of many at least a polymer motion, described many barriers are positioned to a series of row, each described row perpendicular to described flow direction location and in each row of the row of described series each contiguous barrier to by separately greater than the distance of 50 times of described minimum diameters; With
(c) at least a polymkeric substance in fluid sample, every kind of described polymkeric substance have the diameter more than or equal to minimum diameter,
Wherein said at least a polymkeric substance is moving to the direction of described second end from described first end.
97. the integrating device of claim 96, wherein said at least a polymkeric substance comprise DNA and in each row of the row of described series each adjacent barrier be 100nm-800nm to the described distance of separating.
98. the integrating device of claim 97, wherein each adjacent barrier equals 500nm to the described distance of separating in each row of the row of described series.
99. the integrating device of claim 96, each row of the row of wherein said series has total barrier xsect and total channel width, described total barrier xsect equals along described flow direction by the total area that stops of barrier described in each row of the row of described series, equal the total area that do not stopped along described flow direction with described total channel width, the ratio that wherein said total barrier xsect and described total channel width have 0.5-20 by the described barrier of each row described in the row of described series.
100. the integrating device of claim 99, wherein said at least a polymkeric substance comprise that DNA and described ratio are between 2 and 4.
101. the integrating device of claim 96, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
102. the integrating device of claim 101, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
103. the integrating device of claim 96, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
104. the integrating device of claim 103, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
105. integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises a passage, along the described at least a polymkeric substance of this passage, when existing, move on flow direction and wherein said passage comprises the barrier of many described at least a polymer motion, described many barriers reduce on cross-sectional area along described flow direction.
106. the integrating device of claim 105, wherein said passage is taper.
107. the integrating device of claim 105, it further comprises many lateral access that is connected to described passage.
108. the integrating device of claim 105, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
109. the integrating device of claim 108, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
110. the integrating device of claim 105, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
111. the integrating device of claim 110, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
112. an integrating device that is used to stretch DNA, it comprises a kind of elongated structure,
Wherein said elongated structure comprises the centre gangway of a taper, and the centre gangway of described taper comprises first end and second end and wherein said DNA, when existing, move in direction from described first end to described second end along described tapered center passage,
Wherein said elongation further comprises many lateral access that are connected to described tapered center passage,
Wherein said tapered center passage comprises at least one bending; With
Wherein said tapered center passage comprises the barrier of many described DNA motions.
113. the integrating device of claim 112, wherein said tapered center passage reduces on width to described second end linearly from described first end.
114. the integrating device of claim 112, wherein said tapered center passage reduces with the speed greater than linearity on width to described second end from described first end.
115. the integrating device of claim 112, at least one of wherein said many lateral access is taper.
116. the integrating device of claim 112, the equipment of the described DNA of wherein said conveying comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
117. the integrating device of claim 112, wherein said tapered center passage has sinusoidal shape.
118. the integrating device of claim 112, wherein said tapered center passage has flexuose shape.
119. the integrating device of claim 112, wherein said many barriers reduce at cross-sectional area to described second end from described first end.
120. the integrating device of claim 112, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
121. the integrating device of claim 120, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
122. the integrating device of claim 112, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
123. the integrating device of claim 122, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
124. an integrating device that is used to stretch DNA, it comprises a kind of elongated structure, and described elongated structure comprises:
(a) one first tapered channel, described first tapered channel comprises one first end, second end and staggered many pillars between described first end and described second end, it constitutes 12-15 row, the width of described first tapered channel reduces with 26.6 ° angle, described angle at described first end with respect to a constant-width channel definition, described first end has the width between 0.5 and 5 μ m, and described pillar has and equals 1.5 μ m
2Cross-sectional area and the gap that is equaled 0.5 μ m separate; With
(b) one second tapered channel, described second tapered channel is connected to described first tapered channel and width is decreased between the 0.5 and 5 μ m at described second end, therefore a shearing force that produces constant shear rate is applied to described DNA, when it exists, described second tapered channel have 1 and 3mm between length.
125. the integrating device of claim 124, the width of wherein said second tapered channel satisfies equation: W=(SHx/Q+C)
-1, wherein W is described width, and S is described shear rate, and Q is that total flow and C is constant.
126. the integrating device of claim 125, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
127. the integrating device of claim 126, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
128. the integrating device of claim 125, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
129. the integrating device of claim 128, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
130. a method that is used to stretch at least a polymkeric substance, it may further comprise the steps:
Described at least a polymkeric substance is moved along a kind of elongated structure, and described elongated structure comprises a tapered channel with first end and one second end;
Described whereby tapered channel causes producing the shearing force of constant shear rate, when described at least a polymkeric substance during to described second end motion, is applied to described at least a polymkeric substance along described tapered channel from described first end.
131. the integrating device of claim 130, the width of wherein said tapered channel satisfies equation: W=(SHx/Q+C)
-1, wherein W is described width, and S is described shear rate, and Q is that total flow and C is constant.
132. the method for claim 131 wherein saidly makes described at least a polymkeric substance realize by means of capillarity along the step that described tapered channel moves.
133. the method for claim 131, the wherein said step that described at least a polymkeric substance is moved along described tapered channel realizes by means of forming concentration gradient along described tapered channel.
134. the method for claim 131, the wherein said step that described at least a polymkeric substance is moved along described tapered channel are to form pressure head by means of described first end at described tapered channel to realize.
135. the method for claim 134, wherein said pressure head are to form by means of described first end that syringe pump is connected to described tapered channel.
136. the method for claim 135, wherein said elongated structure further comprise a bypass that is connected to described tapered channel.
137. the method for claim 131, the wherein said step that described at least a polymkeric substance is moved along described tapered channel are to form pressure drop by means of described second end at described tapered channel to realize.
138. the method for claim 131 wherein saidly makes step that described at least a polymkeric substance moves along described tapered channel by means of realizing along described tapered channel formation temperature gradient.
139. the method for claim 131, wherein said at least a polymkeric substance comprise that charged polymkeric substance and the described step that described at least a polymkeric substance is moved along described tapered channel realize by means of forming electric field along described tapered channel.
140. the method for claim 139, wherein said electric field have 1000 and 2000V/m between field intensity.
141. two charged on the contrary electrodes that the method for claim 139, wherein said electric field are used in the solution form.
142. the method for claim 139, wherein said electric field forms with a series of electrodes in solution.
143. a method that is used to stretch at least a polymkeric substance, it may further comprise the steps:
At least a polymkeric substance is moved along a kind of elongated structure, and described elongated structure comprises a passage with convergent linearly of first end and second end,
Wherein said at least a polymkeric substance along described passage from described first end to described second end motion.
144. a method that is used to stretch at least a polymkeric substance, it may further comprise the steps:
At least a polymkeric substance is moved along a kind of elongated structure, and described elongated structure comprises a tapered channel with first end and second end, and described tapered channel reduces with the speed greater than linearity to described second end from described first end,
Wherein said at least a polymkeric substance along described passage from described first end to described second end motion.
145. a method that is used to stretch at least a polymkeric substance, it may further comprise the steps:
At least a polymkeric substance is moved along a kind of elongated structure, and described elongated structure comprises a centre gangway that holds fluid and many holds the lateral access that fluid is connected to described centre gangway, and described centre gangway comprises first end and second end,
Wherein said at least a polymkeric substance along described centre gangway from described first end to described second end motion.
146. the method for claim 145 wherein saidly makes described at least a polymkeric substance realize by means of capillarity along the step that described centre gangway moves.
147. the method for claim 145, the wherein said step that described at least a polymkeric substance is moved along described centre gangway realizes by means of forming concentration gradient along described centre gangway.
148. the method for claim 145, the wherein said step that described at least a polymkeric substance is moved along described centre gangway are to form pressure head by means of described first end at described centre gangway to realize.
149. the method for claim 148, wherein said pressure head are to form by means of described first end that syringe pump is connected to described centre gangway.
150. the method for claim 149, wherein said elongated structure further comprise a bypass that is connected to described centre gangway.
151. the method for claim 145, the wherein said step that described at least a polymkeric substance is moved along described centre gangway are to form pressure drop by means of described second end at described centre gangway to realize.
152. the method for claim 145 wherein saidly makes step that described at least a polymkeric substance moves along described centre gangway by means of realizing along described centre gangway formation temperature gradient.
153. the method for claim 145, wherein said at least a polymkeric substance comprise that charged polymkeric substance and the described step that described at least a polymkeric substance is moved along described centre gangway realize by means of forming electric field along described centre gangway.
154. the method for claim 153, wherein said electric field have 1000 and 2000V/m between field intensity.
155. two charged on the contrary electrodes that the method for claim 153, wherein said electric field are used in the solution form.
156. the method for claim 153, wherein said electric field forms with a series of electrodes in solution.
157. the method for claim 145, wherein said centre gangway is taper.
158. the method for claim 145, at least one of wherein said lateral access are tapers.
159. a method that is used to stretch at least a polymkeric substance, it may further comprise the steps:
Described at least a polymkeric substance is moved along a kind of elongated structure, described elongated structure comprises that has at least one crooked passage, described passage comprises first end and second end, wherein said at least a polymkeric substance from described first end to described second end motion
Wherein said at least a polymkeric substance along described passage from described first end to described second end motion.
160. the method for claim 159 wherein saidly makes described at least a polymkeric substance realize by means of capillarity along the step that described passage moves.
161. the method for claim 159, the wherein said step that described at least a polymkeric substance is moved along described passage realizes by means of forming concentration gradient along described passage.
162. the method for claim 159, the wherein said step that described at least a polymkeric substance is moved along described passage are to form pressure head by means of first end at described passage to realize.
163. the method for claim 162, wherein said pressure head are to form by means of described first end that syringe pump is connected to described passage.
164. the method for claim 163, wherein said elongated structure further comprise a bypass that is connected to described passage.
165. the method for claim 159, the wherein said step that described at least a polymkeric substance is moved along described passage are to form pressure drop by means of second end at described passage to realize.
166. the method for claim 159, the wherein said step that described at least a polymkeric substance is moved along described passage realizes by means of forming thermograde along described passage.
167. the method for claim 159, wherein said at least a polymkeric substance comprise that charged polymkeric substance and the described step that described at least a polymkeric substance is moved along described passage realize by means of forming electric field along described passage.
168. the method for claim 167, wherein said electric field have 1000 and 2000V/m between field intensity.
169. two charged on the contrary electrodes that the method for claim 167, wherein said electric field are used in the solution form.
170. the method for claim 167, wherein said electric field forms with a series of electrodes in solution.
171. the method for claim 159, wherein said centre gangway is taper.
172. the method for claim 159, wherein said elongated structure further comprise many lateral access that is connected to described passage.
173. the method for claim 159, wherein said passage has sinusoidal shape.
174. the method for claim 159, wherein said passage has flexuose shape.
175. a method that is used to stretch at least a polymkeric substance, it may further comprise the steps:
At least a polymkeric substance is moved along a kind of elongated structure, described elongated structure comprise a passage and in described passage the barrier of many described at least a polymer motion, described centre gangway comprises one first end and one second end;
Wherein said at least a polymkeric substance along described passage from described first end to described second end motion.
176. the method for claim 175 wherein saidly makes described at least a polymkeric substance realize by means of capillarity along the step that described passage moves.
177. the method for claim 175, the wherein said step that described at least a polymkeric substance is moved along described passage realizes by means of forming concentration gradient along described passage.
178. the method for claim 175, the wherein said step that described at least a polymkeric substance is moved along described passage are to form pressure head by means of first end at described passage to realize.
179. the method for claim 178, wherein said pressure head are to form by means of described first end that syringe pump is connected to described passage.
180. the method for claim 179, wherein said elongated structure further comprise a bypass that is connected to described passage.
181. the method for claim 175, the wherein said step that described at least a polymkeric substance is moved along described passage are to form pressure drop by means of second end at described passage to realize.
182. the method for claim 175, the wherein said step that described at least a polymkeric substance is moved along described passage realizes by means of forming thermograde along described passage.
183. the method for claim 175, wherein said at least a polymkeric substance comprise that charged polymkeric substance and the described step that described at least a polymkeric substance is moved along described passage realize by means of forming electric field along described passage.
184. the method for claim 183, wherein said electric field have 1000 and 2000V/m between field intensity.
185. two charged on the contrary electrodes that the method for claim 183, wherein said electric field are used in the solution form.
186. the method for claim 183, wherein said electric field forms with a series of electrodes in solution.
187. the method for claim 175, wherein said passage is taper.
188. the method for claim 175, wherein said elongated structure further comprise many lateral access that is connected to described centre gangway.
189. the method for claim 175, wherein said passage comprises at least one bending.
190. the method for claim 189, wherein said passage has sinusoidal shape.
191. the method for claim 189, wherein said passage has flexuose shape.
192. the method for claim 175, wherein said many barriers reduce to described second end from described first end along described passage on cross-sectional area.
193. a method that is used to stretch at least a polymkeric substance, it may further comprise the steps:
At least a polymkeric substance is moved along a kind of elongated structure, described elongated structure comprise a passage and in described passage the barrier of many described at least a polymer motion, described centre gangway comprises one first end and one second end;
At least one of wherein said barrier have non-tetragonal polygonal shape of cross section and wherein said at least a polymkeric substance along described passage from described first end to described second end motion.
194. the method for claim 193 wherein saidly makes described at least a polymkeric substance realize by means of capillarity along the step that described passage moves.
195. the method for claim 193, the wherein said step that described at least a polymkeric substance is moved along described passage realizes by means of forming concentration gradient along described passage.
196. the method for claim 193, the wherein said step that described at least a polymkeric substance is moved along described passage are to form pressure head by means of first end at described passage to realize.
197. the method for claim 196, wherein said pressure head are to form by means of described first end that syringe pump is connected to described passage.
198. the method for claim 197, wherein said elongated structure further comprise a bypass that is connected to described passage.
199. the method for claim 193, the wherein said step that described at least a polymkeric substance is moved along described passage are to form pressure drop by means of second end at described passage to realize.
200. the method for claim 193, the wherein said step that described at least a polymkeric substance is moved along described passage realizes by means of forming thermograde along described passage.
201. the method for claim 193, wherein said at least a polymkeric substance comprise that charged polymkeric substance and the described step that described at least a polymkeric substance is moved along described passage realize by means of forming electric field along described passage.
202. the method for claim 201, wherein said electric field have 1000 and 2000V/m between field intensity.
203. two charged on the contrary electrodes that the method for claim 201, wherein said electric field are used in the solution form.
204. the method for claim 201, wherein said electric field forms with a series of electrodes in solution.
205. the method for claim 193, wherein said passage is taper.
206. the method for claim 193, wherein said elongated structure further comprise many lateral access that is connected to described centre gangway.
207. the method for claim 193, wherein said passage comprises at least one bending.
208. the method for claim 207, wherein said passage has sinusoidal shape.
209. the method for claim 207, wherein said passage has flexuose shape.
210. the method for claim 193, wherein said many barriers reduce to described second end from described first end along described passage on cross-sectional area.
211. a method that is used to stretch at least a polymkeric substance, it may further comprise the steps:
At least a polymkeric substance is moved along a kind of elongated structure, and described elongated structure comprises:
(i) one has at least one crooked tapered center passage, and described tapered center passage comprises one first end and one second end;
(ii) many lateral access that is connected to described tapered center passage; With
The (iii) barrier of the many described at least a polymer motion in described tapered center passage;
Wherein said at least a polymkeric substance along described centre gangway from described first end to described second end motion.
212. the method for claim 211 wherein saidly makes described at least a polymkeric substance realize by means of capillarity along the step that described centre gangway moves.
213. the method for claim 211, the wherein said step that described at least a polymkeric substance is moved along described centre gangway realizes by means of forming concentration gradient along described centre gangway.
214. the method for claim 211, the wherein said step that described at least a polymkeric substance is moved along described centre gangway are to form pressure head by means of described first end at described centre gangway to realize.
215. the method for claim 214, wherein said pressure head are to form by means of described first end that syringe pump is connected to described centre gangway.
216. the method for claim 215, wherein said elongated structure further comprises the bypass that is connected to described centre gangway.
217. the method for claim 211, the wherein said step that described at least a polymkeric substance is moved along described centre gangway are to form pressure drop by means of described second end at described centre gangway to realize.
218. the method for claim 211 wherein saidly makes step that described at least a polymkeric substance moves along described centre gangway by means of realizing along described centre gangway formation temperature gradient.
219. the method for claim 211, wherein said at least a polymkeric substance comprise that charged polymkeric substance and the described step that described at least a polymkeric substance is moved along described centre gangway realize by means of forming electric field along described centre gangway.
220. the method for claim 219, wherein said electric field have 1000 and 2000V/m between field intensity.
221. two charged on the contrary electrodes that the method for claim 219, wherein said electric field are used in the solution form.
222. the method for claim 219, wherein said electric field forms with a series of electrodes in solution.
223. the method for claim 211, wherein said tapered center passage has sinusoidal shape.
224. the method for claim 211, wherein said tapered center passage has flexuose shape.
225. the method for claim 211, wherein said many barriers reduce to described second end from described first end on cross-sectional area.
226. claim 130,143,144,145,159,175,193 or 211 method, it carries the step of described polymkeric substance to described elongated structure before further being included in described movement step.
227. the integrating device of claim 11 or 16, wherein said tapered channel on width with 1/ (ax
n+ b) speed reduces to described second end from described first end, and wherein n is the real number greater than 1, and a is the real number of non-zero, and b is that real number and x are the distances along the length of described passage.
228. the integrating device of claim 227, wherein n is an integer.
229. the integrating device of claim 228, wherein the value of n is 2,3 or 4.
230. claim 10,16,27,42,62 or 96 integrating device, wherein said fluid sample further comprises viscosity-modified component.
231. the integrating device of claim 230, wherein said viscosity-modified component is selected from glycerine, sucrose, wood sugar, D-sorbite, polyglycol, polyacrylamide and polyethylene oxide.
232. the integrating device of claim 230, wherein said viscosity-modified component comprises 4 ℃ aqueous buffer solutions.
233. integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, wherein said elongated structure comprises a passage, along the described at least a polymkeric substance of this passage, when existing, on flow direction, move and wherein said passage comprises that at least one reduces the height of described passage to second end from first end, z, step.
234. the integrating device of claim 233, wherein said passage have the length from 1 μ m to 1mm.
235. the integrating device of claim 233, wherein said at least one step has the height from 0.1 μ m to 0.9 μ m.
236. the integrating device of claim 233, wherein said passage reduces on width to described second end linearly from described first end.
237. the integrating device of claim 233, wherein said passage on width with 1/ (ax
n+ b) speed reduces to described second end from described first end, and wherein n is the real number greater than 1, and a is the real number of non-zero, and b is that real number and x are the distances along the length of described passage.
238. the integrating device of claim 233, wherein said at least one step makes the height of described passage, and z is decreased to 2 to 1/100th.
239. the integrating device of claim 233, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
240. the integrating device of claim 239, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
241. the integrating device of claim 233, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
242. the integrating device of claim 241, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
243. an integrating device, it comprises:
A kind of elongated structure that comprises a passage, described passage comprises at least one step, it makes the height of described passage, z, reduce from first end to the second end, described passage comprises at least a polymkeric substance in fluid sample, described passage be arranged so that described at least a polymkeric substance from described first end when the direction of described second end is moved, apply a shearing force thereon.
244. the integrating device of claim 243, wherein said passage have the length from 1 μ m to 1mm.
245. the integrating device of claim 243, wherein said at least one step has the height of from 0.1 to 0.9 μ m.
246. the integrating device of claim 243, wherein said passage reduces on width to described second end linearly from described first end.
247. the integrating device of claim 243, wherein said passage on width with 1/ (ax
n+ b) speed reduces to described second end from described first end, and wherein n is the real number greater than 1, and a is the real number of non-zero, and b is that real number and x are the distances along the length of described passage.
248. the integrating device of claim 243, wherein said fluid sample further comprises a kind of viscosity-modified component.
249. the integrating device of claim 243, wherein said at least one step makes the height of described passage, and z is decreased to 2 to 1/100th.
250. an integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, and described elongated structure comprises:
(a) first passage, described first passage comprise one first end and one second end; With
(b) second channel, described second channel comprise one the 3rd end and one the 4th end, and described the 3rd end is connected to described first passage at described second end,
Along the described at least a polymkeric substance of passage, when existing, on flow direction, move and
Wherein said first passage reduces at width from described first end to described second end, and its speed that reduces is different from the speed that described second channel reduces at width to described the 4th end from described the 3rd end.
251. the integrating device of claim 250, wherein said first passage and described second channel have the length from 1 μ m to 1mm independently of one another.
252. the integrating device of claim 250, wherein said first passage further comprise many pillars between described first end and described second end.
253. the integrating device of claim 252, at least one of wherein said many pillar has non-tetragonal polygonal shape of cross section.
254. the integrating device of claim 252, at least one of wherein said many pillar has the oval cross section shape, and the major axis of wherein said oval cross section shape is perpendicular to described flow direction.
255. the integrating device of claim 252, at least one of wherein said many pillar has rectangular cross-sectional shape, and the major axis of wherein said rectangular cross-sectional shape is perpendicular to described flow direction.
256. the integrating device of claim 252, at least one of wherein said many pillar comprises the edge of spill, the direction that the edge facing fluid of wherein said spill comes.
257. the integrating device of claim 252, wherein each pillar in described many pillars has at 0.1 μ m
2With 10 μ m
2Between cross-sectional area, described many pillars are positioned in a series of row between 12 to 15.
258. the integrating device of claim 257, wherein the packing factor of six organ timbering increases to 50% from 0% on described flow direction, the distance at center that wherein said at least 12 to 15 rows subsequently have constant 50% packing factor and wherein said at least 12 to 15 rows' adjacent rows equals 2 μ m.
259. the integrating device of claim 257, wherein said pillar row's packing factor be from 0% to 80% increase continuously on described flow direction.
260. the integrating device of claim 257, wherein said pillar row's packing factor is constant.
261. the integrating device of claim 252, the cross-sectional area of wherein said many pillar along described flow direction from 10 μ m
2To 1 μ m
2Reduce.
262. the integrating device of claim 250, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
263. the integrating device of claim 262, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
264. the integrating device of claim 250, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
265. the integrating device of claim 264, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
266. an integrating device that is used for stretching at least a polymkeric substance at fluid sample, it comprises a kind of elongated structure, and described elongated structure comprises:
(a) first passage, its width equals 10 μ m and highly equals 1 μ m, described first passage comprises one first end, second end and staggered many pillars between described first end and described second end, it constitutes at least 12 to 15 rows, described many pillars finish at described second end, and each pillar in described many pillar has 1-25 μ m
2Cross-sectional area; With
(b) second channel, described second channel comprise one the 3rd end and one the 4th end, and described the 3rd end is connected to described first passage at described second end, described second channel on width from described the 3rd end to described the 4th end with 1/x
2Speed dwindle, described beam overall narrows down to 1 μ m from 10 μ m, wherein x is the distance along the length of described second channel, the length of described second channel equals 5 μ m, described second channel is included in height to the 0.25 μ m that described the 3rd end reduces described second channel
2A step,
Wherein said at least a polymkeric substance when existing, moves on flow direction along described first passage and described second channel.
267. the integrating device of claim 266, at least one of wherein said many pillar has non-tetragonal polygon shape of cross section.
268. the integrating device of claim 266, at least one of wherein said many pillar has the oval cross section shape, and the major axis of wherein said oval cross section shape is perpendicular to described flow direction.
269. the integrating device of claim 266, at least one of wherein said many pillar has rectangular cross-sectional shape, and the major axis of wherein said rectangular cross-sectional shape is perpendicular to described flow direction.
270. the integrating device of claim 266, at least one of wherein said many pillar comprises the edge of spill, and the edge surface of wherein said spill is to described flow direction.
271. having, the integrating device of claim 266, each of wherein said many pillar equal 1 μ m
2Cross-sectional area.
272. the integrating device of claim 271, wherein the packing factor of six organ timbering increases to 50% from 0% on described flow direction, the distance at center that wherein said at least 12 to 15 rows subsequently have constant 50% packing factor and wherein said at least 12 to 15 rows' adjacent rows equals 2 μ m.
273. the integrating device of claim 271, the packing factor of wherein said at least 12 to 15 rows' pillar be from 0% to 80% increase continuously on described flow direction.
274. the integrating device of claim 266, the cross-sectional area of wherein said many pillar on described flow direction from 10 μ m
2To 1 μ m
2Reduce.
275. the integrating device of claim 266, it further comprises at least one detection zone in described second channel.
276. the integrating device of claim 266, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
277. the integrating device of claim 276, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
278. the integrating device of claim 266, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
279. the integrating device of claim 278, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
280. the integrating device of claim 266, wherein said fluid sample further comprises a kind of viscosity-modified component.
281. one kind is used for length is the integrating device that at least a polymkeric substance at fluid sample is stretched on the basis selectively, it comprises a kind of elongated structure, and wherein said elongated structure comprises:
(a) first passage, described first passage comprise one first end, second end and between described first end and described second end staggered many pillars, each pillar is located in described second end of distance and is not less than L in described many pillars; With
(b) second channel, described second channel comprise one the 3rd end and one the 4th end, and described the 3rd end is connected to described first passage at described second end, and described second channel dwindles at width to described the 4th end from described the 3rd end,
Along the described at least a polymkeric substance of passage, when existing, on flow direction, move.
282. one kind is used for stretching many integrating devices at the polymkeric substance of fluid sample with different length, it comprises a kind of elongated structure, and wherein said elongated structure comprises:
(a) first passage, described first passage comprise one first end and one second end;
(b) second channel, described second channel comprise one the 3rd end and one the 4th end, and described the 3rd end is connected to described first passage at described second end, and described second channel dwindles at width to described the 4th end from described the 3rd end; With
(c) staggered many pillars in described first passage and described second channel,
Along the described many polymkeric substance of passage, when existing, on flow direction, move.
283. the integrating device of claim 281 or 282, wherein said first passage and described second channel have the length from 1 μ m to 1mm independently of one another.
284. the integrating device of claim 281 or 282, it further comprises at least one detection zone in described second channel.
285. the integrating device of claim 281 or 282, at least one of wherein said many pillar has non-tetragonal polygon shape of cross section.
286. the integrating device of claim 281 or 282, at least one of wherein said many pillar has the oval cross section shape, and the major axis of wherein said oval cross section shape is perpendicular to described flow direction.
287. the integrating device of claim 281 or 282, at least one of wherein said many pillar has rectangular cross-sectional shape, and the major axis of wherein said rectangular cross-sectional shape is perpendicular to described flow direction.
288. the integrating device of claim 281 or 282, at least one of wherein said many pillar comprises the edge of spill, and the edge surface of wherein said spill is to described flow direction.
289. the integrating device of claim 281 or 282, at least one of wherein said many pillar has at 0.1 μ m
2With 10 μ m
2Between cross-sectional area.
290. the integrating device of claim 281 or 282, wherein said second channel reduces on width to described the 4th end linearly from described the 3rd end.
291. the integrating device of claim 281 or 282, wherein said second channel on width with 1/ (ax
n+ b) speed reduces to described second end from described first end, and wherein n is the real number greater than 1, and a is the real number of non-zero, and b is that real number and x are the distances along the length of described second channel.
292. the integrating device of claim 281, it further comprises the equipment of the described at least a polymkeric substance of conveying in described fluid sample to described elongated structure.
293. the integrating device of claim 282, it further comprises carries described many polymkeric substance in described fluid sample to arrive the equipment of described elongated structure.
294. the integrating device of claim 292 or 293, wherein said conveying equipment comprises transfer passage, and described elongated structure is introduced and drawn to described transfer passage.
295. the integrating device of claim 281, it further comprises makes described at least a polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
296. the integrating device of claim 282, it further comprises makes described many polymkeric substance, when existing, and the equipment that in described elongated structure, moves.
297. the integrating device of claim 295 or 296, wherein said equipment comprise the equipment that forms pressure differential along described elongated structure.
298. claim 233,243,250,266,281 or 282 integrating device, it further comprises at least a polymkeric substance in fluid sample.
299. the method for claim 144, wherein said passage on width with 1/ (ax
n+ b) speed reduces to described second end from described first end, and wherein n is the real number greater than 1, and a is the real number of non-zero, and b is that real number and x are the distances along the length of described passage.
300. claim 130,131,143 or 144 method, wherein said passage further comprises at least one step, and it makes the height of described passage, and z reduces to described second end from described first end.
301. further comprising, the method for claim 300, wherein said method carry described at least a polymkeric substance to described elongated structure.
302. the method for claim 300, the wherein said step that described at least a polymkeric substance is moved along described passage are by means of forming pressure head at described passage first end, forming vacuum or it is in conjunction with realizing by means of second end at described passage.
303. a method that is used to stretch at least a polymkeric substance, it comprises:
Described at least a polymkeric substance is moved along a kind of elongated structure, and described elongated structure comprises a first passage, and described first passage comprises one first end and one second end; With a second channel, described second channel comprises one the 3rd end and one the 4th end, described the 3rd end is connected to described first passage at described second end, wherein said first passage reduces at width from described first end to described second end, and its speed that reduces is different from the speed that described second channel reduces at width to described the 4th end from described the 3rd end.
304. the method for claim 303, it further comprises carries the step of described at least a polymkeric substance to described elongated structure.
305. the method for claim 303, wherein said first passage further comprise many pillars between described first end and described second end.
306. the method for claim 303, the wherein said step that described at least a polymkeric substance is moved along described first passage and described second channel are to form pressure head, form vacuum or it is in conjunction with realizing by means of the 4th end at described second channel by means of first end at described first passage.
307. the method for claim 302 or 306, wherein said pressure head are to form by means of first end that syringe pump is connected to described first passage.
308. one kind be used for stretching at least a fluid sample, length is more than or equal to the method for the polymkeric substance of L, it comprises:
Described at least a polymkeric substance is moved along a kind of elongated structure, described elongated structure comprises a first passage, described first passage comprises one first end, one second end and between described first end and described second end staggered many pillars, it is L that each pillar in described many pillars is located in described second end of distance, with a second channel, described second channel comprises one the 3rd end and one the 4th end, described the 3rd end is connected to described first passage at described second end, described second channel dwindles at width to described the 4th end from described the 3rd end
Have wherein that polymkeric substance more than or equal to the length of L stretches and the polymkeric substance that has less than the length of L is not stretched.
309. the method for claim 308, it further comprises carries the step of described at least a polymkeric substance to described elongated structure.
310. the method for claim 308, the wherein said step that described at least a polymkeric substance is moved along described first passage and described second channel are to form pressure head, form vacuum or it is in conjunction with realizing by means of the 4th end at described second channel by means of first end at described first passage.
311. one kind is used for stretching many methods at the polymkeric substance of fluid sample with different length, it comprises:
Described many polymkeric substance are moved along a kind of elongated structure, and described elongated structure comprises:
(a) first passage, described first passage comprise one first end and one second end;
(b) second channel, described second channel comprise one the 3rd end and one the 4th end, and described the 3rd end is connected to described first passage at described second end, and described second channel dwindles at width to described the 4th end from described the 3rd end; With
(c) staggered many pillars in described first passage and described second channel.
312. the method for claim 311, it further comprises carries described many polymkeric substance to arrive the step of described elongated structure.
313. the method for claim 311, the wherein said step that described many polymkeric substance is moved along described first passage and described second channel are to form pressure head, form vacuum or it is in conjunction with realizing by means of the 4th end at described second channel by means of first end at described first passage.
314. the method for claim 310 or 313, wherein said pressure head are to form by means of first end that syringe pump is connected to described first passage.
315. a method that is used to stretch at least a polymkeric substance, it comprises:
Described at least a polymkeric substance is moved along a kind of elongated structure, and described elongated structure comprises:
(a) width equals 10 μ m and highly equals the first passage of 1 μ m, described first passage comprises one first end, second end and staggered many pillars between described first end and described second end, it constitutes at least 12 to 15 rows, and described many pillars have 1-25 μ m in described second end end and each pillar in described many pillars
2Cross-sectional area; With
(b) second channel, described second channel comprise one the 3rd end and one the 4th end, and described the 3rd end is connected to described first passage at described second end, described second channel from described the 3rd end to described the 4th end on width with 1/x
2Speed dwindle, described beam overall is contracted to 1 μ m from 10 μ m, and wherein x is the distance along the length of described second channel, and the length of described second channel equals 5 μ m, described second channel comprises a step at described the 3rd end, and it makes the height of described second channel be reduced to 0.25 μ m
2
316. the method for claim 315, it carries the step of described polymkeric substance to described elongated structure before further being included in described movement step.
317. the method for claim 143 or 144 wherein saidly makes described at least a polymkeric substance realize by means of capillarity along the step that described tapered channel moves.
318. the method for claim 143 or 144, the wherein said step that described at least a polymkeric substance is moved along described tapered channel realizes by means of forming concentration gradient along described tapered channel.
319. the method for claim 143 or 144, the wherein said step that described at least a polymkeric substance is moved along described tapered channel are to form pressure head by means of described first end at described tapered channel to realize.
320. the method for claim 319, wherein said pressure head are to form by means of described first end that syringe pump is connected to described tapered channel.
321. the method for claim 320, wherein said elongated structure further comprise a bypass that is connected to described tapered channel.
322. the method for claim 143 or 144, the wherein said step that described at least a polymkeric substance is moved along described tapered channel are to form pressure drop by means of described second end at described tapered channel to realize.
323. the method for claim 143 or 144 wherein saidly makes step that described at least a polymkeric substance moves along described tapered channel by means of realizing along described tapered channel formation temperature gradient.
324. the method for claim 143 or 144, wherein said at least a polymkeric substance comprises that charged polymkeric substance and the described step that described at least a polymkeric substance is moved along described tapered channel realize by means of forming electric field along described tapered channel.
325. the method for claim 324, wherein said electric field have 1000 and 2000V/m between field intensity.
326. two charged on the contrary electrodes that the method for claim 324, wherein said electric field are used in the solution form.
327. the method for claim 324, wherein said electric field forms with a series of electrodes in solution.
328. claim 1,10,11,16,17,27,43,60,62,63,70,75,83,89,96 or 105 integrating device, it further comprises at least one detection zone in described passage.
329. claim 28,40,41,42 or 112 integrating device, it further comprises at least one detection zone in described centre gangway.
330. the integrating device of claim 124, it further comprises at least one detection zone in described second channel.
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|---|---|---|---|
| US14902099P | 1999-08-13 | 1999-08-13 | |
| US60/149,020 | 1999-08-13 |
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|---|---|
| CN1379857A true CN1379857A (en) | 2002-11-13 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN00814266A Pending CN1379857A (en) | 1999-08-13 | 2000-08-11 | Method and apparatus for stretching polymers |
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| Country | Link |
|---|---|
| EP (1) | EP1210578A4 (en) |
| JP (1) | JP2003507026A (en) |
| CN (1) | CN1379857A (en) |
| AU (1) | AU6771100A (en) |
| CA (1) | CA2381361A1 (en) |
| IL (1) | IL148088A0 (en) |
| WO (1) | WO2001013088A1 (en) |
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| US8722327B2 (en) | 2007-03-28 | 2014-05-13 | Bionano Genomics, Inc. | Methods of macromolecular analysis using nanochannel arrays |
| CN102369059B (en) * | 2008-06-06 | 2014-09-24 | 博纳基因技术有限公司 | Integrated analysis devices and related fabrication methods and analysis techniques |
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- 2000-08-11 WO PCT/US2000/022253 patent/WO2001013088A1/en active Application Filing
- 2000-08-11 AU AU67711/00A patent/AU6771100A/en not_active Abandoned
- 2000-08-11 JP JP2001517140A patent/JP2003507026A/en active Pending
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Also Published As
| Publication number | Publication date |
|---|---|
| AU6771100A (en) | 2001-03-13 |
| EP1210578A4 (en) | 2007-02-21 |
| WO2001013088A1 (en) | 2001-02-22 |
| EP1210578A1 (en) | 2002-06-05 |
| CA2381361A1 (en) | 2001-02-22 |
| JP2003507026A (en) | 2003-02-25 |
| IL148088A0 (en) | 2002-09-12 |
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