CN101080500A - Nanopores, methods for using same, methods for making same and methods for characterizing biomolecules using same - Google Patents

Nanopores, methods for using same, methods for making same and methods for characterizing biomolecules using same Download PDF

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CN101080500A
CN101080500A CN 200480011566 CN200480011566A CN101080500A CN 101080500 A CN101080500 A CN 101080500A CN 200480011566 CN200480011566 CN 200480011566 CN 200480011566 A CN200480011566 A CN 200480011566A CN 101080500 A CN101080500 A CN 101080500A
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hole
nanoporous
insulating element
rna
crystal
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X·S·凌
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Brown University
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Abstract

The present invention relates to a device or system for detecting or characterizing the single macromolecule or measuring the sequence of the DNA or RNA or confirming the one or a plurality of solid nanometer pores of the secondary structure of the RNA. In one solid nanometer pore of the invention, the breadth or length of the nanometer pore is limited or confirmed by the shape edge of the chapped crystal, the shape edge remain stationary relationship at the course when the insulation components including nanometer pore are formed. On the other aspect of the invention, it relates to the linear or 2-D electricity-addressable nanometer pore array, wherein, the nanometer pore lies at the intersection between the groove formed at the upper surface of the insulation component and the groove formed at the lower surface of the insulation component.

Description

Nanoporous uses the method for nanoporous, prepares the method for nanoporous and with the method for nanoporous characterising biological molecule
Invention field
The device that the present invention relates to be used for the characterising biological molecule and DNA and RNA are checked order, more particularly, the nanoporous (nanopore) and/or the nanohole array that the present invention relates to be used for the fast characterizing biomolecules He carry out the high-throughput dna sequencing.
Background of invention
Is ubiquitous by the nucleic acid of nano level membrane channel and the transhipment of other biological credit at nature.Example comprises the motion by nucleopore of RNA molecule and transcription factor; The viral DNA of in phage-infect, injecting by bacterial film; With the picked-up of membranin to specific oligonucleotides.How understand the function of described nano level passage and biomolecules is modern biomolecular science and biophysical main task by their motions.In recent years, at molecular biology, the biophysical fascinating field of nanoporous had appearred in the interdisciplinary zone between nanotechnology (nanotechnology) and the single molecular biophysics.In 1994, Berzrukov, Vodyanoy and Parsegian showed that people can be with the biology nanoporous as Ku Leerte particle (Coulter) counter, with the counting individual molecule.In 1996, at Kasianowicz, Brandin, [Kasianowicz, L J, Brandin, E, Branton, D.﹠amp in the epoch-making paper of Branton and Deamer (KBBD); Deamer, D.W.Characterization of individual polynucleotide molecules using amembrane channel, Proc.Nat.Acad.Sci.USA93,13770-13773 (1996)] propose to utilize the nanoporous ionic conductance to carry out the design ingenious of the single molecule order-checking of ultra-high speed of strand (ss) dna molecular as testing mechanism.The marquis rises thenceforth, and several research groups had been explored already with the potentiality of alpha hemolysin albumen hole as the possible material standed for of realizing this purpose.
According to the KBBD method, utilize the nanoporous ionic conductance to carry out dna sequencing, relate to two insulating walls that container separates with solion, wherein, described wall comprises so little hole, to such an extent as to have only one single strand dna can be fit to by described hole.When these two containers were applied voltage, current potential reduced and should almost all occur on the nanoporous.And the situation that some contact resistance between two blocks of metals or electricity are led is similar, and the electricity between two ion acceptors is led or resistance is that the electricity of described nanoporous is led or resistance equally.Because the harmonic motion of described dna molecular, the ionic conductance by described nanoporous is by the Na by described nanoporous +And Cl -Flow decision.When dna molecular entered described nanoporous, described ionic conductance reduced.
The amount that electricity is led reduction is the measuring of physical size of the dna molecular part in the narrowest part of passage.Because the chemical group (Nucleotide) of dna molecular has different slightly sizes, so people can measure the genetic information of the Nucleotide of dna molecular by the time-dependent manner of measuring the nanometer pore conductance.
The potential hypothesis of described KBBD method is that four kinds of different Nucleotide that constitute DNA or RNA molecule may have different blocking effects in ion(ic)current when described molecule moves by nanoporous, form because they have different atoms.Figure 1 illustrates voltage and drive the key concept of DNA by the nanoporous transhipment, show among the figure that (a) uses the voltage of alpha hemolysin nanoporous to drive the synoptic diagram of DNA transport experiment, (b) at damping fluid 1M KCl, use the general transhipment incident of poly-deoxyadenylic acid among the 1mM Tris-EDTA (pH 8.5).
The challenging preliminary data that had obtained already shows that this design is feasible.For example, the homopolymer (DNA) that had shown poly--VITAMIN B4 is already compared with poly-cytosine(Cyt) and has been produced more lower slightly blocking effect, and is with the speed transhipment of the some microseconds of each base at ambient temperature.Above-mentioned discovery is very challenging for realizing the rapid DNA sequence measurement really.
The utilization of described KBBD method is embedded in alpha hemolysin protein ion passage in the lipid bilayer as the natural nano hole.Confirmed already in described nanometer pore conductance, to have produced measurable signal really by the DNA polymkeric substance that 70 cytidylic acid(CMP)s and 30 adenine nucleotides are formed.But, because the natural nano hole is long passage, normal length is 30nm, so a lot of any time of Nucleotide in transport process all is present in the described passage, and has lost the influence of single nucleotide pair ionic conductance.People are C and the difference of A Nucleotide in the nanometer pore conductance as can be seen, this fact is the omen that people finally can see the influence of single nucleotide pair nanometer pore conductance, if people can prepare the length nanoporous suitable with single Nucleotide (approximately 0.4nm).
Confirmed also that recently (Golovchenko ' s group in the Harvard PhysicsDepartment) is used for forming reliable nanometer engraving (nano sculpting) method that diameter is the single nanoporous of 1.5nm on silicon-nitride solid film.In the method, the treatment step that uses focused ion beam imprint lithography and have a low-yield sputter of feedback monitoring is highly reproducible and reliable, but, for the single Nucleotide of measurement, the length of this nanoporous still oversize (＞10nm).J.Li, D Stein, C McMullan, DBranton, M.J.Aziz and J.A.Golovchenko; Ion-beam sculpting atnanometer length scales, Nature 412,166-169 (2001).
In the experiment that Meller etc. carries out recently, find that current blocking amount and dna molecular have big fluctuation by the time that the nanoporous passage is spent.A.Meller, L.Nivon and DBranton, Voltage-driven DNA translocations through a nanopore, Phys.Rev.Lett.86,3435-3438 (2001).Figure 2 illustrates expression blocking-up electric current to the transport speed of polymkeric substance length (Image to left) and same polymer histogram to polymkeric substance length (Image to right).Therefore, the good understanding to the origin of this fluctuation is necessary as can be seen, so that observe single base in the standard DNA transport experiment.In fact, for linear flexible molecule (as DNA) how under the influence of motivating force the physical understanding by nanoporous also seldom make that to go out size information from the ion(ic)current extracting data very difficult.There are some theory literatures that all respects of this problem are studied, as the influence (entropy barrier) of the fluctuation of polymer ends, DNA-passage interaction etc.From the experiment side, measuring the DNA transport process with constant speed (if possible under low-speed conditions) or constant power is the height ideal.The temperature dependent form meter of the DNA transhipment of being undertaken by the alpha hemolysin hole in the past understands that the thermal motion of DNA is the source of noise in the blocking-up electric current.People are devoted to stop transport process always, and DNA is remained in the alpha hemolysin hole.
It is desirable to, people wish to measure ion(ic)current, make dna molecular full extension (for example using optical tweezers (referring to Fig. 3)) in described hole simultaneously.In this experiment, because the fluctuation of the ion(ic)current that the DNA thermal motion causes can be weakened or eliminate.In addition, people can keep molecule static, and use signal averaging, so that further reduce the noise in the ion(ic)current.But, for this reason, people need (a) with single stranded DNA attached on the double-stranded DNA, then with the latter attached to (for example, using streptavidin-vitamin H joint) on the pearl, fix [referring to the introduction of optical tweezers by optical tweezers; Www.nbi.dk/～tweezer/introduction.htm], (b) drives described single stranded DNA by described hole then, and catches its (using streptavidin-vitamin H joint equally) with another pearl from opposite side.Except the optics trapping, above-mentioned all processes all are at random, and may expend time in very much.In order to make this design feasible, people need use high-throughput device, and as nanohole array, and each hole on described array must be electrically addressable, and is independently.
At present, two kinds of successful method that are used for making the solid nano hole on insulating material are arranged.A kind of method relates to the ionic fluid engraving that utilizes silicon nitride, and another kind of method is used a-beam imprint lithography, and carries out wet etching in crystalline form silicon, carries out oxidation then.Generally, already described technology had been used to confirm that nanoporous can use in the manner described, had had high-throughout solid nano aperture apparatus but also be not used in to produce.
USP 6,428, and 959 have described the method for measuring the existence of double-strandednucleic acid in sample.In described method, the nucleic acid that is present in the fluid sample is transported by nanoporous, for example, and by described fluid sample is applied electric field.In transport process, monitoring is by the current amplitude of described nanoporous, and the change of amplitude is relevant by the passage of described nanoporous with strand or duplex molecule.Described method can be applicable in the multiple use, wherein, need to detect the existence of double-strandednucleic acid in sample, for example, in hybridization assays, measures as the RNA trace, and southern blotting technique is measured, based on the hybridization assays of array etc.
May need to provide the novel nano hole that can use synthetic materials to form, and the method for using and prepare described nanoporous.May also need to provide the nanoporous of length≤10nm, the apparatus and method of producing nanoporous linear array or nanoporous two-dimensional array need be provided.Such linearity and two-dimensional nano hole array also need be provided especially, and wherein, described nanoporous is to distinguish electrically addressable.
Summary of the invention
The present invention relates to comprise the device and the system in one or more solid nanos hole, described nanoporous can be used for detecting and/or characterizing single macromole, and DNA or RNA are checked order.Described device and system serve many purposes at molecular biology and single molecular biophysics field.Except can be used for the rapid DNA order-checking, device of the present invention and system can be used in the multiple use, include, but are not limited to single molecular biophysics, molecular biology and biological chemistry.For example, expect that nanoporous device of the present invention and system can be used as molecular comb (molecular comb), be used to survey the secondary structure of RNA molecule, be used for the contaminants/pollutent in detection of biological war reagent and air and/or the water.
According to an aspect of the present invention, relate to the device of the parts that comprise insulating material, wherein, described insulating element is designed and arranges,, comprise nanoporous therein so that comprise through hole.Described through hole comprises a plurality of crystal, and these crystal carried out cracking, so that form sharp edge atomically, described crystal is arranged with the fixed relation when forming described insulating material.In specific embodiments, described crystal edge intersects each other with predetermined angle, more particularly with about 90 angle of intersection of spending.But, for described crystal edge each other with less than or greater than the angle of intersection of 90 degree, so that form the nanoporous with varying cross-section or varying cross-section shape, this within the scope of the invention.
The described crossing crystal edge that splits constitutes basically or defines enough little position, makes the molecule of forming described insulating material can not enter this position.Like this, the limit orientation that the molecule of described insulating material should describedly relatively split, and produce certain profile around therefore forcing in described point of crossing, thus be created in aperture comprising described through hole.In specific embodiments, described crystal is the GaAs crystal, and this crystal is well-known in the art.But, the present invention can use other crystal known in those skilled in the art to implement, and has similar feature, described feature makes them to be split and concerns maintenance with fixed, formed the described insulating material that comprises described insulating element simultaneously, for example, the NaCl crystal.
In specific embodiments, described insulating material is the material with following feature: it is flowable, and when forming the end of processing of described nanoporous and during normal operating condition (for example, room temperature), can solidify.In the embodiment of the property illustrated, described insulating element is made with the solidifiable polymkeric substance, and as with liquid PDMS (polydimethylsiloxane), polystyrene or PMMA and GaAs crystal are used to form described nanoporous through hole.This technology has obtained length or passage length is 20  (dust/10 -10M) or littler nanoporous, more particularly passage length is 20  or littler, or more particularly passage length is about 4 , and more specifically passage length (d) can satisfy one of following relational expression: 2 ≤d≤10  or 4 ≤d≤10 .
In described illustrative example, the GaAs crystal that splits (semiconductor substrate materials commonly used) is so that form sharp edge atomically.Arrange the described crystal that splits then, so that two such crystal edges are put together, they are in the distance of several dusts, and be maintained fixed with standard STM electronic installation, and utilize the electron tunnel electric current as Feedback mechanism (this supposition GaAs crystal is doped, and has limited electroconductibility).In a more particular embodiment, the spacing of described crystal edge is 1 -10 , more specifically is about 2 .
In other embodiments, described crystal edge separates each other, so that the distance between the crystal edge is less than the molecular thickness (Tm) of described insulating material.In a more particular embodiment, the spacing between the described crystal edge (Se) is configured to satisfy following relation: Se/Tm≤0.5.
Then solidifiable polymeric liquid PDMS (polydimethylsiloxane) is cast to described cutting edge (cutting edge) zone, and solidifies, simultaneously two crystal edges are fixed together.Pointed as this paper, on the point of crossing between two crystal edges, the distance between the described limit is so little, to such an extent as to the molecule of polymkeric substance can not enter this zone.Described polymer molecule equally advantageously is parallel to the limit orientation of splitting separately, and forces to form certain profile around in the point of crossing, produces aperture.In this method, the width of nanoporous and length are to control by the diameter of distance between two limits and polymkeric substance.
After solidifying or forming the final form of described insulating element, remove described crystal, for example, by washing.In another kind of exemplary, the crystal that is used to form described through hole is the NaCl crystal, and described crystal is removed by washing with water.
Also relate to and utilize nanoporous device of the present invention to carry out any one system and method in the various analysis, described analytical procedure includes, but are not limited to the characterising biological molecule, and DNA is checked order and definite RNA secondary structure.Described method comprises the insulating element that provides mentioned above, it comprises nanoporous, wherein, the diameter of described nanoporous and length are to be determined by the crystalline sharp edge that splits, they are maintained fixed relation during forming described insulating element, and locate described insulating element, so that it is deployed between two ion acceptors.Described method also comprises allows described biomolecules or DNA pass through from described nanoporous, and characterizes described biomolecules or DNA according to the change of ion(ic)current or other physics parameters.For the method for determining the RNA secondary structure, this method comprises also that operationally the end with RNA is coupled on the optical tweezers, and measures the power of a described end when described RNA molecule is pulled through described nanoporous.
According to a further aspect in the invention, relate to the nanohole array of electrically addressable, it is designed and arranges can carrying out the high throughput analysis of biomolecules, and DNA is checked order.In a kind of specific embodiments, the nanohole array of described electrically addressable comprises linearity or 1-dimention nano hole array.In another embodiment, the nanohole array of described electrically addressable comprises the nanoporous two-dimensional array.In other embodiments, described two-dimensional array is a plurality of linear array forms.In other embodiments, described linearity and two-dimensional nano hole array form in described insulating material and arrange, so that each nanoporous can use, and for example electric current addressing independently that provides by the standard patch clamp.
In specific embodiments, the nanohole array of described electrically addressable comprises insulation material layer.Described insulating element is designed and arranges, so that it is had at its first surface upper edge first direction longitudinal extension and one or more grooves parallel to each other.Described insulation material layer is designed and arranges, so that also formed groove at its second surface upper edge second direction longitudinal extension on the second surface of described insulation material layer, wherein, described first and second surfaces are respect to one another.Equally, the relative first direction of described second direction forms certain angle, and more particularly, described first and second directions are perpendicular to one another, or form about an angle of 90 degrees each other.
Also formed groove on each in first and second surfaces,, thereby on the point of crossing of each groove on the first surface and the groove on the second surface, formed the opening that comprises nanoporous so that extend downwardly into another surface from a surface.Like this, the linearity or the one-dimensional array of a plurality of nanoporouss on described insulating material have been formed on the point of crossing of the groove that forms on each in a plurality of grooves on first surface and the second surface.
As indicated above, in other embodiments, described insulation material layer is designed and arranges, so that on its second first surface, have along a plurality of grooves second direction longitudinal extension and parallel to each other.Also on described second surface, formed a plurality of grooves,, thereby on the point of crossing of each groove on the first surface and each groove on the second surface, formed the opening that comprises nanoporous so that extend downwardly into another surface from a surface.Like this, on each point of crossing of a plurality of grooves on first surface and the second surface, in described insulating material, formed the two-dimensional array of a plurality of nanoporouss.In a more particular embodiment, described groove is a V-shape.
In exemplary, described insulation material layer is formed by first subgrade and second subgrade, they be by employing well known to a person skilled in the art in the multiple technologies any one in conjunction with or fixed, described technology is fit to employed material, so that form described insulation material layer.Described first subgrade is designed and arranges, so that comprise a plurality of grooves by extending between the apparent surface, one of described facing surfaces comprises the first surface on described insulating material surface.Similarly, second subgrade is designed and arranges, so that be included in the groove that extends between the apparent surface, one of described apparent surface comprises the second surface on described insulating material surface.In other embodiments, and pointed as this paper, second subgrade can be designed and arrange, so that comprise a plurality of grooves that each extends between the apparent surface, one of described apparent surface comprises the second surface on described insulating material surface.When being bonded to each other or be fixed together, first subgrade and second subgrade are directed, so that the first surface of described insulation material layer and second surface are respect to one another, thereby the one or more flute profiles on the groove on first subgrade and second subgrade are angled, more particularly, described first subgrade and second subgrade are directed, so that the groove in subgrade separately is perpendicular to one another or forms an angle of 90 degrees each other.
In specific embodiments, the top end part at least of each groove on first subgrade and second subgrade is designed and arranges, being open, and each groove is to form in first subgrade and second subgrade each, so that separated from one another.Equally described open portion is arranged, so that on each point of crossing of each groove on first subgrade and the one or more grooves on second subgrade, formed the opening that comprises nanoporous.Like this, on the point of crossing of each groove of each and second subgrade of a plurality of grooves on first subgrade, on described insulating material, formed the linearity or the one-dimensional array of a plurality of nanoporouss.In a more particular embodiment, described groove is a V-shape, and the top end part of described V-shape groove by moulding being open.
Exemplary, in the illustrative embodiment, first subgrade and second subgrade comprise silicon chip and use the oxide compound or the nitride mask that are dissolved in the KOH solution to form groove on described silicon chip.By control etching speed and etching period, described technology makes it possible to form groove, more especially V-shape groove with high precision (for example, in 20nm).After on described silicon chip surface, etching groove, first subgrade and second subgrade are combined.After combination, this assembly is carried out oxidation, so that form SiO 2, so that isolate all silicon faces.
The system and method that also relates to the nanohole array that adopts electrically addressable of the present invention is used for carrying out any one of various analysis, and described analytical procedure includes, but are not limited to the characterising biological molecule, and DNA is checked order and definite RNA secondary structure.Described method comprises the nanohole array of the linear or two-dimentional electrically addressable that provides as indicated above, and described insulating element is positioned, so that be deployed between two ion acceptors.Described method also comprises allows described biomolecules or DNA pass through from any one or a plurality of nanoporous of described linearity or two-dimensional array, and characterizes described biomolecules or DNA according to the change of ion(ic)current or other physics parameters.For the method for determining the RNA secondary structure, described method comprises that also the end with RNA operationally is coupled on the optical tweezers, and measures the power on a described end when the RNA molecule is pulled through any one nanoporous that constitutes described linearity or two-dimensional array.
According to a further aspect in the invention, relate to the nanoporous device of PNA functionalization, and the method and system relevant with it.Described PNA functionalization comprise the nanoporous device, it comprises one or more nanoporous described herein and PNA coating, adopting this coating is in order to apply the internal surface of one or more at least nanoporouss at least.In a more particular embodiment, described PNA material is a synthetic, so that characterized or constituted by one of base of forming DNA or RNA molecule.Relevant method comprises that the end with the DNA/RNA molecule operationally is coupled on the optical tweezers, and measures the power on the described end when described molecule is pulled through any one nanoporous that constitutes described linearity or two-dimensional array.Described method also comprises described DNA/RNA molecule is checked order, this is by measuring the fluctuation as the power of the function of time, and make that base of described fluctuation and the described PNA coating of sign and appear at DNA or RNA on multiple base in any one between related realization of keying action.
Other aspects of the present invention and embodiment are discussed below.
The accompanying drawing summary
Character for a more complete understanding of the present invention and required purpose, with reference to the following detailed description of carrying out in conjunction with the accompanying drawings, wherein, similarly reference number is represented corresponding part in all several views, wherein:
Fig. 1 represents the key concept of the DNA of voltage driving by the nanoporous transhipment, more particularly (a) is to use the synoptic diagram of the DNA transport experiment that the voltage of alpha hemolysin nanoporous drives, (b) the general transhipment incident of expression, its use is present in damping fluid 1M KCl, the poly-deoxyadenylic acid among the 1mM Tris-EDTA (pH 8.5);
Fig. 2 A, B provide the blocking-up electric current to polymkeric substance length (Fig. 2 A) and with the histogram of transport speed and the polymkeric substance length (Fig. 2 B) of same polymer;
Fig. 3 is the sketch that expression utilizes the cutting edge mechanism of solidifiable polymer formation nanoporous;
Fig. 4 is the synoptic diagram of the exemplary Feedback mechanism of the distance between two cutting edges of control;
Fig. 5 is the 3-D view of the part of solidified polymeric after removing the GaAs crystal;
Fig. 6 is the skeleton view of the nanohole array of electrically addressable according to an aspect of the present invention;
Fig. 7 is according to the skeleton view of the nanohole array of the electrically addressable with nanoporous linear array of the present invention, for the sake of clarity, has only a groove above the device at this;
Fig. 8 is the synoptic diagram according to the nanohole array of the electrically addressable with nanoporous two-dimensional array of the present invention;
Fig. 8 A is the top view of the part section (cutaway) of the two-dimensional array that is made of a plurality of single nanoporous assemblies of the present invention;
Fig. 8 B is the side-view of array shown in Fig. 8 A by a nanoporous assembly;
Fig. 9 is that expression uses pearl and optical tweezers to keep the molecule synoptic diagram by nanoporous still;
Figure 10 is the sketch of the expression property the illustrated optics and the general arrangement of the nanohole array device of video system and their relative electrically addressables of the present invention;
Figure 11 is the synoptic diagram of the technology of the expression secondary structure information that is used to obtain relevant RNA; With
Figure 12 A, B are the synoptic diagram of the sequencing technologies of the expression nanoporous that uses PNA functionalization of the present invention.
The description of preferred embodiment
Referring to each figure in the accompanying drawing, wherein, similarly reference number is represented similar parts, and Fig. 3-5 shows the various views that explanation is used to prepare the method for nanoporous insulating element 10, and according to an aspect of the present invention, described parts comprise nanoporous 12.More particularly, Fig. 3 is the sketch of the cutting edge mechanism of the expression nanoporous 12 that uses solidifiable polymer formation insulating element 10 of the present invention; Fig. 4 is the synoptic diagram of the exemplary Feedback mechanism of the distance between two cutting edges of expression control crystalline; With Fig. 5 be the 3-D view of the part of described insulating element 10 after the GaAs crystal is removed.
According to an aspect of the present invention, provide nm probe (nanopore) insulating element 10, it is specialized designs and arrangement, so that comprise nanoporous 12 therein, described hole is that the thickness that passes described parts extends.In a more particular embodiment, described insulating element is to be made by in the multiple insulating material known in those skilled in the art any one, and is suitable for described insulating element of the present invention.In use, described insulating nano probe component 10 is installed, so that the container of two solions is separated, wherein, described insulating element comprises the hole, and promptly nanoporous 12, determines the size of described nanoporous so that preferably have only one single strand dna therefrom to pass through.Equally, when on these two containers, applying voltage, help on nanoporous, producing current potential fully and reduce.
Below explanation is used to prepare a kind of technology of insulating element 10 of the present invention.But, expection additive method or technology also are fit to keep crystal to be in fixed relationship, so that the mechanism that limits the through hole that comprises nanoporous is provided.This method advantageously produces the insulating element with nanoporous, can control the length or the passage length of described parts, is about 20  (dust/10 -10M) or littler, more particularly passage length is about 10  or littler, and more particularly passage length is about 4 , and passage length (d) satisfies one of following relation more specifically: 2 ≤d≤10  or 4 ≤d≤10 .Pointed as this paper, the routine techniques that is used to form the nanoporous of this purposes has obtained following structure, and wherein the length of nanoporous has surpassed the length of the DNA chain that will characterize, and this may cause the inaccuracy that characterizes.
As illustrated in this technology, the crystal that splits so that form sharp edge atomically, and is put together two in these crystal that split and to be made them each other in the distance of several dusts.In addition, described crystal is arranged, so that crystal edge is tangent with the angle of about 90 degree each other, referring to the diagram of Fig. 3.Utilize standard STM (scanning tunnel microscope) electronic installation as shown in Figure 4, and utilize the electron tunnel electric current, keep two crystalline fixed relationships as Feedback mechanism.On the point of crossing between two limits, the distance between the limit is so little, can not enter this position to such an extent as to surpass the molecule of certain size.In other words, set up described point of crossing, can not enter this position so that width surpasses the molecule of required width.In a more particular embodiment, the crystalline limit separates each other, so that in 1 -10  scope, and more specifically about 2 .In other embodiments, described crystal edge is separated from one another, so that the distance between the crystal edge is less than the thickness (Tm) of the molecule of described insulating material.In a more particular embodiment, set the spacing (Se) between the described crystal edge, so that satisfy following relation: Se/Tm≤0.5.
The crystal that expection is used for the present invention comprises multiple crystal, and concrete, in the exemplary illustrative embodiment, described crystal comprises the GaAs crystal, and it is common semiconductor material and NaCl crystal.But, the present invention can use other crystal enforcements known in those skilled in the art and that have similar characteristics, and these materials self can be split them, and are maintained fixed relation when forming the described insulating material that constitutes described insulating element.
Described crystal uses the crystal edge that splits that intersects as mould (for example, nanometer molding (nano-molding)) when arranging like this.After arranging crystal mutually with the fixed relation, another kind of insulating material (for example, flowable insulating material), cutting edge position as described in being injected into as solidifiable polymeric liquid PDMS (polydimethylsiloxane), allow them to solidify, keep the fixing of two crystal edges simultaneously.Determine the distance between the described limit, make them enough little, so that there is not molecule can enter this forbidden zone.Therefore, the molecule that comprises the described insulating material of forming insulating element 10 can not enter so-called forbidden zone.In Fig. 3, also show a kind of possible molecular conformation near the polymkeric substance in described point of crossing.The molecule of described insulating material advantageously is parallel to the limit orientation of splitting separately, and is forced to form the profile around described point of crossing, forms the aperture with specific width and length.Control the width and the length of described aperture by the diameter of distance between two limits and described insulating material (for example, polymkeric substance).
In other embodiments, and after insulating element 10 moulding like this, employing well known to a person skilled in the art that in the multiple technologies any one removed the crystal that is used to controllably form nanoporous or opening on described insulating element, this technology can not influence the described insulating material in the relevant hole that forms significantly, for example, for the NaCl crystal, by washing with water.After removing described crystal, on described parts, formed opening, as shown in Figure 5, it has width and the length that needs.Because the chemical group (Nucleotide) of dna molecular has slightly different size, thus the Nucleotide that people can the identification of dna molecule, and therefore come dna molecular is checked order by the time-dependent manner of measuring the nanometer pore conductance.Like this, on described insulating material, formed nanoporous; It has width and the length that needs for the purpose that characterizes described biomolecules, particularly is fit to the described biomolecules of fast characterizing.Therefore, such insulating element 10 has the multi-purpose big purposes of being fit to, and comprises medical jurisprudence, rapid DNA order-checking and research.Pointed as this paper, the amount that electricity is led reduction is to be positioned at described passage the measuring of physics size of the part of the dna molecular of narrow part.
Referring to Fig. 6, wherein show the skeleton view of device 100, it comprises the nanohole array 112 of electrically addressable, nanoporous linear array especially according to a further aspect in the invention.Device 100 comprises the insulating element 110 with upper surface 112 and lower surface 114.Employing well known to a person skilled in the art that in the multiple technologies any one formed a plurality of grooves 116, the material of the described insulating element of the suitable formation of described technology on described upper surface.Groove 116 on the upper surface 112 forms on insulating element 110, so that generally extend predetermined distance from described upper surface downwards towards lower surface 114.Correspondingly, on lower surface 114, forming groove 118 on the insulating element 110, so that generally extend upward predetermined distance towards upper surface 112 from described lower surface.
In addition, on the groove 116 and the point of crossing between the groove 118 in the lower surface 114 in upper surface 112, described insulating element is designed and arranges, so that on described insulating element 110, form opening or nanoporous 122, so that make upper surface groove 116 and lower surface channels 118 fluid coupling each other.To discuss the formation of groove 116,118 and nanoporous 122 below in more detail.
In a more particular embodiment, insulating element 110 comprises the first layer 120a and second layer 120b, they are to adopt to well known to a person skilled in the art that in the multiple technologies any one is bonding or otherwise be fixed together, and described technology is suitable for forming the material of described insulating element.In this specific embodiments, upper surface groove 116 is included on the first layer 120, and lower surface channels is included on the second layer.
In the use multiple technologies any one, on each of described insulating element or first and second layers, formed nanoporous 122, described technology is fit to employed material and is fit to form the nanoporous with required width and length, for example to be used for the fast characterizing biomolecules.In a kind of specific embodiments, with first and second layers of 120a, b in conjunction with or be fixed together, and on the point of crossing of upper surface and lower surface channels 116,118, form nanoporous 116.In another kind of specific embodiments, upper surface and lower surface channels 116 have been formed, 118 tip portion, so that comprising an opening near in each groove in point of crossing, thereby as first and second layers of 120a, b in conjunction with or when being fixed together, the sharp edge opening on described groove align so that the formation nanoporous.
In addition, in shown embodiment, in upper surface and lower surface 112,114, formed groove 116,118, so that dark V-shape groove is provided.This shape is not restrictive, because as known in those skilled in the art, be fit to and can be used among the present invention with other shapes expections that the function of groove of the present invention is coincide yet.
In the embodiment that specifically illustrates, insulating element 110 and first and second layers are made with silicon materials at first, and after described insulating element was further processed, shape was carried out oxidation to it then, become SiO 2, it is an insulating material.Although described starting material are silicon, this should not be considered to be determinate, because the expection other materials can be used for the present invention, this allows to prepare nanoporous 122 linear arraies described herein with the technology that is fit to employed material.
More particularly, described silicon materials are processed, to form groove 116,188 and nanoporous 122 with micromachining technology known in those skilled in the art.Specifically, use oxide compound or nitride mask, in KOH solution,, perhaps pass through to use beam imprint lithography and wet etch techniques etching with high relatively tolerance range etching bath and nanoporous on described silicon chip.Have found that routine known in the field or standard wet etch process can be produced the uniform groove of height as shown in Figure 6.
In other embodiments, to form first and second layers of 120a, the silicon chip of b in conjunction with or be fixed together, use then to be similar to and form the used method of groove being positioned at the point of crossing of upper surface groove 116 and lower surface channels 118, be that material on the tip of described groove is done further etching, so that form through hole there.In another embodiment, during forming groove 116,118, the tip of described groove or sunk part, the part in the point of crossing of particularly close upper surface and lower surface channels is carried out further etching, so that form opening at these tip portions.First and second layers are bonded to each other or are fixed together, and the sharp edge of described opening has formed nanoporous on described point of crossing.Like this, formed " cutting edge " hole, a kind of way is by two silicon chips are combined, the sharp edge that makes described groove with an angle of 90 degrees toward each other, another kind of way is by from the direct etching in the one or both sides of described silicon chip, its groove is relative with an angle of 90 degrees each other.As known in those skilled in the art,, can accurately control etching to described surface by control etching speed and etching period.In described etching with in conjunction with constituting first and second layers of 120a, after the silicon chip or material of b, this assembly is oxidized to SiO 2, so that isolate all silicon faces, thus but the insulating element of formation operation format.
In other embodiments, and forming insulating element 110, but, using and further check nanoporous 122, with the width of determining that whether formed nanoporous has needs such as electron microscope so that it is in after the operation format.If not, and the width of supposing specific nanoporous then carries out high-power electron beam to described nanoporous and handles, with the size of change nanoporous less than critical width.Describe more comprehensively as following, when the contact electron beam, aperture can be because surface tension spontaneously be shunk, and after turning off electron beam, described material quenches and keeps its shape.
Further specify this improving one's methods below, described method is for example used, commercialization transmission electron microscope (TEM), and it is worked under the acceleration voltage of 300kV.Well-known in electron microscopy is that high electronics intensity may be damaged sample or make the sample distortion, and in general, people attempt to make this effect to minimize.But, in the present invention, this effect is used to controllably change the size of silicon oxide nano pore.Have found that about 10 6-10 7A/m 2Electronics intensity may cause the hole to be shunk, if the green diameter in described hole is about 50nm or littler.
The effectiveness of this technology is the possibility with the diameter of unprecedented accurate way fine setting nanoporous.After reaching the diameter that needs already,, can in seconds stop described contraction process by reducing intensity of beam or eliminating beam.In addition, can adopt microscopical image-forming mechanism to monitor the change of bore dia in real time.By improving electronics intensity, can make contraction roughly accelerate at least one order of magnitude, and can weaken gradually for final control.Precision finally is subjected to microscopical resolution limit.In practice, described resolving power is confined to about 1nm, and this is because the surfaceness of silicon oxide.The controlled levels that provides by this technology is at least than the high order of magnitude of conventional e-beam imprint lithography, and the latter's final resolving power is about 10nm.Therefore, the use of this improvement technology provides the mechanism that weakens the size control that needs in the imprint lithography that is used to form groove and nanoporous, because any diameter all may be retracted to the hole of nanometer size less than the hole of 50nm.
With the physical structure of using observed growth of above-mentioned electron beam process and contraction already is measurement of surface tension by the viscosity silicon oxide.In this state, described structure can be out of shape becomes the configuration with lower free energy F.Simple free energy factor has shown that the surface energy in the hole of radius r＜ h can reduce by dwindling r, and the surface energy in the hole of radius r＞ h can improve by increasing size." critical diameter " of differentiating these two kinds of situations on the order of magnitude of the thickness of described silicon, definite ratio depends on the geometrical shape in described hole.This scaling all is effectively on any rank, and can explain viewed kinetics in our hole well.
The advantage of technology of the present invention is, uses the direct vision feedback on inferior nano-resolution level, and it is feasible that nanometric sample is modified.The measured silicon of described process is handled and can be by the TEM microscopy of commercial channel acquisition.In the lithography that limits described hole, the appropriate resolution of＜50nm is essential, because as final step, electron beam is finely tuned.Use is based on the method for SOI, and this requirement is for realizing it being simple with e-beam imprint lithography, and even only just should realize with the optics imprint lithography.Compare with " ionic fluid engraving " technology of nearest report, other advantages of the technology of the present invention are the chemical constitutions that technology of the present invention can not change hole material on every side, because described electron beam has softened glassy silicon oxide, make it to be out of shape and to drive lentamente by surface tension.Electron microscope also provides the real-time vision feedback, and, when having obtained the morphology of needs already, reduce electron beam intensity, and with SiO 2Be quenched into its pristine glass shape state.
Referring to Fig. 8, wherein show the synoptic diagram of the nanohole array 200 of the electrically addressable of the present invention that constitutes by nanoporous two-dimensional array 116.In embodiment illustrated in fig. 6, two-dimensional array is made up of the two or more groups linear array, and described array forms on an insulating element 110.In shown embodiment, on upper surface, formed groove 116a, b so that there is one group of groove 116a to be used for one of described linear array, and has another to organize each that groove 116b is used for other linear arraies.In other embodiments, formed groove 116a on upper surface 112 or the first layer 120a, b is so that the groove of the groove of each linear array and another linear array is isolating or is not connected.For example, when described groove is on the upper surface at silicon chip during etching, advantageously do not remove the material on the upper surface between the described groove two ends.
It is restrictive that above content should not be considered to, because following situation within the scope of the invention, promptly the groove 116 in the two-dimensional array upper surface of the present invention is designed,, thereby form one group of upper surface groove so that the groove in described upper surface is interconnective.For example, for analyzing or characterizing under the unessential situation, as mentioned below being used to analyzed under the situation of RNA secondary structure, can design described two-dimensional array, so that the groove in the upper surface is interconnective, so that form one group of groove at ion(ic)current.
Referring to Fig. 7, wherein show the skeleton view of the nanohole array device 300 of electrically addressable of the present invention, it comprises the nanohole array 100 of electrically addressable, also comprises nanoporous linear array 122.For the sake of clarity, only illustrate or illustrated a upper surface groove 116.But, this should not be regarded as determinate, and is a plurality of because described linear array 100 can comprise, for example, 100 upper surface grooves, thus 100 nanoporouss formed.In addition, although show linear array, described device comprises that the two-dimensional array 200 that contains array shown in Figure 8 also belongs to scope of the present invention.
In above-mentioned discussion, should be with reference to figure 6 and 8, there is shown other details of array 100,200 of electrically addressable that expection is used for the nanohole array device 300 of electrically addressable of the present invention.Equally, in order to describe, show electrode and fluid port.In actual device, described electrode and fluid port seal.
As known in those skilled in the art, the nanohole array device 300 of electrically addressable of the present invention comprises cover slip 302, seals the upper surface and the lower surface of described array by it.After the nanohole array 100 of producing described electrically addressable, adopt in the multiple technologies that are fit to employed material known in those skilled in the art any one that cover slip 302 is fixing or be combined on its upper surface and lower surface.This is preferably determining the nanohole array 100 of described electrically addressable, but 200 (for example, the having the width of suitable special analysis Technology Need and the nanoporous of length) of carrying out after being under the operational condition.In addition, as known in those skilled in the art, the nanohole array device 300 of described electrically addressable is suitably to arrange with pumping line and electrode (for example, Ag/AgCl lead).As known in those skilled in the art, other devices, the nanohole array device 300 of equipment and system's (not shown) and electrically addressable interconnects, so that sampling is used for analyzing, is used to collect data, and is used to analyze the data of collecting.
Referring to Fig. 8 A, B wherein shows the various views of two-dimensional nano hole array 500 according to a further aspect in the invention.This two-dimensional array 500 on the one hand according to the present invention, and, comprise a plurality of single nanoporous assembly 510 of the present invention referring to Fig. 8 A, they comprise the nanoporous 512 that one of preferred use technology described herein forms separately.In Fig. 8 A, more specifically show the top view of the part section of this two-dimensional array 500 on the one hand, and the side-view that passes through a nanoporous assembly of array shown in Fig. 8 A has been shown in Fig. 8 B according to the present invention.Has the technology of the nanoporous assembly 512 of the present invention of required feature about preparation, referring to above about the discussion of Fig. 3-5.Also can the structure and the feature of the nanohole array of electrically addressable no longer be described below about further details referring to the discussion of above carrying out in conjunction with Fig. 7.Except nanoporous assembly 510, two-dimensional nano hole array 500 comprises top component 520 and bottom part 530.
In two-dimensional array 500, nanoporous assembly 510 is designed and arranges, so that first space 514 of each nanoporous assembly is isolating and is electrical isolation each other.Top component 520 is added by this way or is fixed on the upper surface of each nanoporous assembly 510, so that can also keep first space, the 514 separated from one another and electrical isolations of each nanoporous assembly.Top component 520 also comprises a plurality of through holes 522, and they are arranged on the described top component, so that there is a hole to be communicated with first space, 514 fluids of each nanoporous assembly 510.
Similarly, bottom part 530 is fixed on the lower surface of each nanoporous assembly 510, so that the passage 516 on each nanoporous assembly and bottom part defines one or more spaces of passing through the material of nanoporous 512 already that are used to receive, so that can carry out suitable further operation or processing to the material that passes through.
Top component 520, bottom part 530 is to make with in the multiple material known in those skilled in the art any one, described material is fit to the purposes of expection and can be fixed on the surface of described nanoporous assembly (for example, cover slip 320) as described herein.Through hole 522 on the top component 520 is to adopt to well known to a person skilled in the art that in the multiple technologies any one make.The size dimension of through hole 522 and the degree of depth are such, so that make material can be easily pass through from its inside, enter first space 514, and will material during by described through hole the destructive possibility to described material drop to minimum.Should be understood that the size of through hole 522 or diameter needn't be set, so that satisfy the identical required feature of nanoporous 512.
This arrangement and structure have obtained a kind of two-dimensional nano hole array 500, wherein, each nanoporous 512 be with any other nanoporous of forming described array independently, electrically addressable.Therefore, can analyze, the material of handling and/or assessing imports in each nanoporous 512, so that the material that passes through from any one nanoporous (for example, DNA) can be identified uniquely or respectively, characterize, analyze, handle or assess.Described two-dimensional array 500 shows high-throughput, but still has kept providing the ability through details, feature or the characteristic of the material of each nanoporous 512.
Nanoporous assembly 510 can also adopt and well known to a person skilled in the art that in the multiple technologies any one fix, and described technology includes, but are not limited to the machinery and the technology that is adhesively fixed, so that effective assembling of nanoporous assembly can form single structure.For example, can use jointing material that described nanoporous assembly is together fixed to one another.In a more particular embodiment, with top component 520, bottom part 530 and nanoporous assembly 510 fixed to one another being in the same place are so that form single structure.
In shown embodiment, two-dimensional array 500 is made up of 4 * 4 matrixes of nanoporous 512 or nanoporous assembly 510.This is not construed as limiting, because the array of being made up of more or less nanoporous or nanoporous assembly belongs to scope of the present invention.For example, two-dimensional array 500 of the present invention can by 1000 more a plurality of nanoporous 512 or 1000 or still less a nanoporous 512 form (array that for example, comprises 100 * 100 nanoporous matrixes).Although (for example show quadrate array, the nanoporous 512 that has similar number in the x and y direction), this is not construed as limiting, because within the scope of the invention, the nanoporous assembly can be arranged like this, make along the number of the nanoporous of an axle and the number different (for example, 100 * 75 nanohole arrays) of nanoporous along another.Be contemplated that equally, can arrange described nanoporous assembly, so that the number of nanoporous 512 can (for example change along an axle, between 100-70 nanoporous, change), and make and to be different from number (for example, 75 nanoporouss) substantially along the nanoporous of an axle along another the number of nanoporous.
Although above described comprise a plurality of, the two-dimensional array 500 that more particularly comprises a large amount of nanoporous assemblies 512, but utilize technical finesse silicon chip described herein, belong to scope of the present invention so that form the nanoporous two-dimensional array, these arrays each all be and the first space fluid link coupled that forms on silicon chip, and therefore, each first space is separated from each other, so that each nanoporous is independently, electrically addressable.
Pointed as this paper, the method for using ion(ic)current to carry out the rapid DNA order-checking still is unrealized so far, and this method can not be used to distinguish VITAMIN B4 and guanine and cytosine(Cyt) and VitB1.Utilize alpha hemolysin protein nano porose area not belong to the also never success of nearest effort of the Nucleotide of purine (VITAMIN B4 and guanine) and pyrimidine (cytosine(Cyt), VitB1 and uridylic) group.But, device of the present invention has overcome the defective of art methods.For example, in one embodiment, the nanohole array device 300 of electrically addressable of the present invention has been done further design and arrangement so that the attenuated signal noise, these noises be considered to since DNA during by nanoporous thermal motion caused.More particularly, nanohole array device 300 to electrically addressable designs and arranges, make object lens 320 be positioned at the either side of described array apparatus, so that form two optical acquisition devices or two optical tweezers, so that on nanoporous 122, keep dna molecular static.Like this, can weaken greatly by near and/or be arranged in the noise of the ion current signal that oscillating motion caused of the dna molecular of nanoporous.By keeping described molecule to fix, can utilize signal-averaging technique further to weaken the noise of ion(ic)current equally.
By following illustrative example, can understand best and keep molecule static, although within those skilled in the art's ken, can adopt and improve the other technologies of the analysis/sign that is more suitable for carrying out project.With single stranded DNA attached on the double-stranded DNA, utilize then streptavidin-vitamin H joint with the latter attached on the pearl 400, described pearl is by the optical tweezers fixed.Drive single stranded DNA then by nanoporous 122, and utilize streptavidin-vitamin H joint to use another pearl 400 to catch from opposite side equally, described pearl is by the optical tweezers fixed equally.In Fig. 9, schematically show and utilize pearl 400 to keep static by the molecule of nanoporous 122.In the property illustrated embodiment, described pearl 400 or microsphere are polystyrene microsphere body or pearl.
About the nanohole array device 300 (being EANA in the accompanying drawings) of electrically addressable of the present invention, figure 10 illustrates the general arrangement of optics and video system 500 and its parts of the property illustrated.This optics and video system 500 are equipped with optical tweezers and digital video microscopic system, and it is based on Zeiss Axiovert 135 inverted microscopes.In other embodiments, can replace shown Argon ion laser, so that the optical damage to the described biomolecules on the microsphere is minimized with the iraser of based semiconductor.This system comprises two object lens, and the condensing apparatus in the normalized optical microscope is replaced by 100 times of oil-immersion objectives.Equally, luminous can the use by the diode light-source that defocuses of object lens of sample realized.
As this area institute common general knowledge, the sub-field of laser physics is optical acquisition, and optical tweezers is a kind of example of optical acquisition device.The laser beam of high order focusing has catches and fixing ability of being scheduled to the particle (for example, the particle of insulating material) in the magnitude range.This technology makes it possible to research and operation as the particle of atom and molecule (even bigger) and small-sized insulativity spheroid.The summary of relevant optical tweezers can be referring to A.Ashkin " Opticaltrapping and manipulation of neutral particles using lasers " Proc.Natl.Acad.Sci.USA, vol.94, pp.4853-4860, in May, 1997.
As known in the field, long (strand) RNA (Yeast Nucleic Acid) molecule self can be partially folded, forms secondary structure, and this is because the part pairing of complementary base.It is believed that with their primary sequence and compare that the secondary structure of RNA is even more important for the characteristic of described molecule.For example, the multiple function of RNA molecule in cell is from carrying genetic information from DNA (thymus nucleic acid) to albumen synthetic messenger RNA(mRNA) (mRNA), to transfer RNA (tRNA) (tRNA), it is carried to rrna at translate duration with amino acid, main secondary structure from them.The physical technique of exploitation rapid determination RNA secondary structure is the major objective of the emerging field of single molecular biophysics.
In the present invention, the damping fluid flushing that will contain microsphere that RNA adheres to or pearl 400 applies bias voltage by electric field to nanoporous 122 simultaneously by lower surface channels 118.By electric field the RNA molecule is pulled through nanoporous 122 accidentally.People can determine this incident by checking near the localization microsphere or the pearl 400 in hole.Then, close described electric field, so that make RNA form secondary structure.Now, also can be referring to Figure 11, the applied optics tweezers pull away microsphere or pearl 400 lentamente, and when the RNA molecule is pulled through (making progress) nanoporous 122, measure the power on the described pearl.Owing to, the Top Down Break Down of RNA base pair can occur, therefore disclosed the information of the secondary structure of relevant test molecule then in the ingress of nanoporous 122 as the fluctuation of the power of the function of time.Should be pointed out that this situation is different from former RNA and pulls out experiment very much, wherein, RNA pulls out from two ends, and when the situation of the secondary structure of not understanding RNA in advance, deterministic force is difficult to the feature of time locus.The present invention does not need to understand in advance the secondary structure of RNA molecule.
Referring to Figure 12 A, B wherein schematically shows the another kind of technology of utilizing nanoporous of the present invention or nanohole array to characterize (for example order-checking) DNA or RNA.More particularly, described synoptic diagram has illustrated the sequencing technologies of the nanoporous 600 that utilizes PNA functionalization of the present invention.Provide the nanoporous device of the present invention that does not have PNA, the various technology of equipment or assembly and the above-mentioned discussion of device referring to relevant.For the sake of clarity, below discussion and accompanying drawing have been illustrated technology of the present invention and device with reference to single nanoporous.But, following disclosed technology and device are improved, so that use a plurality of nanoporouss of arranging, thereby form linear array or two-dimensional array, this is within the scope of the present invention.
After forming nanoporous 602, administration for peptides nucleic acid (PNA) is so that apply at least a portion of described nanoporous, and for example, the internal surface in coated with nano hole 602 at least is so that form PNA coating 604.PNA coating 604 and the nanoporous 602 that so applies are known as the nanoporous 600 of PNA functionalization in this article.Any DNA/RNA that passes through in the opening 606 of PNA coating 604 contacts from the nanoporous 600 of PNA functionalization on the described nanoporous.In specific embodiments, synthetic PNA is so that it constitutes (for example, adenosine, thymidine, guanine, cytosine(Cyt), uridylic) by one of base of DNA or RNA substantially.More particularly, synthetic described PNA, so that determine to characterize or constitute the base of described PNA, thereby DNA/RNA passing through from the nanoporous 600 of PNA functionalization had remarkably influenced, and particularly depend on the different base that to form described DNA/RNA and have different effects.
Just as described, and particularly referring to Figure 12 B, PNA coating 604 is only to have adenosine base (below be referred to as A-PNA) basically in order to be configured or to be characterized.Therefore, A-PNA has different influence (for example, hydrogen bonded) to the nanoporous 600 of DNA/RNA chain by functionalization, this depends on the particular bases that constitutes DNA, and the base sequence on the DNA/RNA, and the distance between PNA coating 604 and the DNA/RNA chain (for example, bonding distance).For example, between A and T base, there is strong keying action, therefore, the DNA/RNA chain applied relatively large power, thereby hinder the motion of described chain in the nanoporous 600 of functionalization.
Adopt technology known in those skilled in the art, technology as described herein, measure or detected power or other relevant parameters (for example time of mobile specific range) that motion is slowed down, and the parameter to detection/mensuration is assessed, so that determine described parameter whether with T as A-PNA and DNA, A, G or C base in conjunction with the time parameter that shown suitable.In a kind of property illustrated technology, drive the nanoporous 600 of the end of DNA/RNA chain, and use pearl 400 to catch from opposite side with streptavidin-vitamin H joint by the PNA functionalization.With the fixing pearl 400 of optical tweezers known in the field.In the another kind property illustrated embodiment, pearl 400 or microsphere are polystyrene microsphere body or pearl.
Use optical tweezers that microsphere or pearl 400 are pulled away from the nanoporous 600 of PNA functionalization lentamente, and when the DNA/RNA molecule is pulled through the nanoporous of described functionalization, measure the power on the described pearl.Pointed as this paper, the base of forming the DNA/RNA molecule can cause different power to be applied on the described molecule, influences the motion of described molecule by the nanoporous 600 of functionalization.When masterpiece is the function fluctuation of time, can show above-mentioned different effect, therefore disclosed the relevant at least A that constitutes test molecule, T, G, the information of the sequence of C base.
Although adopt buzzword that the preferred embodiments of the invention are described, but this description is only used for illustration purpose, and, should be understood that, under the prerequisite of the spirit or scope that do not exceed following claims, can change and change.
Reference is quoted
All patents disclosed herein, disclosed patent application and other documents are done this paper reference with special receipts of their full text form.
Equivalent
It will be appreciated by those skilled in the art that or can determine a lot of equivalents of specific embodiments of the present invention described herein by normal experiment.These equivalents are regarded as belonging to the scope of following claims.

Claims (52)

1. be used for the device of characterising biological molecule, comprise:
Insulating element, described insulating element is included in the through hole that extends between the apparent surface; With
Wherein, the width of described through hole is by when forming described insulating element, and a plurality of crystal of arranging relatively with fixed relationship are determined each other.
2. device as claimed in claim 1, wherein, when forming described insulating element, described a plurality of crystalline limit intersects each other with predetermined angle, and keeps this structure.
3. device as claimed in claim 2, wherein, when forming described insulating element, described limit forms or defines a position, and this position is enough little, makes the molecule of forming described insulating element can not enter this position.
4. device as claimed in claim 2, wherein, the molecule of described insulating material is a described relatively limit orientation, so that force described insulating material to be formed on profile around the described point of crossing, thereby limits the through hole in the described insulating element.
5. the method for a characterising biological molecule may further comprise the steps:
Insulating element is provided, and described insulating element is included in the through hole that extends between the apparent surface, and wherein, the width of described through hole is by when forming described insulating element, and a plurality of crystal of arranging relatively with fixed relationship are determined each other;
Described insulating element is positioned between the ion acceptor, and wherein at least one ion acceptor comprises the described biomolecules that will characterize; With
Allow described biomolecules from the through hole of the insulating element that provided, pass through.
6. method as claimed in claim 5, further comprising the steps of:
When described biomolecules is passed through described through hole, detect the change of ion(ic)current and ion(ic)current; With
According to the change of detected ion(ic)current and ion(ic)current, characterize described biomolecules.
7. the method for a definite RNA secondary structure may further comprise the steps:
Insulating element is provided, and described insulating element is included in the through hole that extends between the apparent surface, and wherein, the width of described through hole is by when forming described insulating element, and a plurality of crystal of arranging relatively with fixed relationship are determined each other;
Described insulating element is positioned between the ion acceptor, and wherein at least one ion acceptor comprises the described biomolecules that will characterize;
Functionally the end with RNA is coupled on the optical tweezers; With
When being pulled through described through hole, measures described RNA molecule the power of a described end.
8. the nanohole array of electrically addressable comprises:
Insulating material part;
Wherein, described insulation material layer is designed and arranges, so that in its first surface, have a plurality of grooves along the first direction longitudinal extension;
Wherein, described insulating material part is designed and arranges, so that have in its second surface along the groove of second direction longitudinal extension, described first and second surfaces are respect to one another;
Wherein, the relative first direction of described second direction forms certain angle; With
Wherein, described groove is to form in first and second surfaces each, so that formed the opening that comprises nanoporous in the point of crossing of each groove on the first surface and the groove on the second surface.
9. the nanohole array of electrically addressable as claimed in claim 8, wherein, described insulating material part is formed by the first layer and the second layer, wherein, the surface of the first layer is the first surface of insulating material part, wherein, described the first layer is included in a plurality of grooves that form in the first surface of described insulating element, wherein, the surface of the described second layer is the second surface of described insulating material part, and the described second layer is included in the groove that forms in the described insulating element second surface.
10. the nanohole array of electrically addressable as claimed in claim 9, wherein, described first and second layers are bonded to each other or are fixed together, so that form described insulating material part.
11. the nanohole array of electrically addressable as claimed in claim 8 wherein, designs and arranges described insulating material part, so that have a plurality of grooves along the second direction longitudinal extension on its second surface.
12. nanohole array as the electrically addressable of claim 11, wherein, described insulation material layer is designed and arranges, so that on its first surface, have a plurality of grooves of a plurality of groups along the first direction longitudinal extension, wherein, each the group in groove be not with another the group in groove be connected.
13. nanohole array as the electrically addressable of claim 12, wherein, described groove is to form in first surface and second surface each, so that each groove in the first surface of each group groove and one point of crossing of a plurality of grooves on second surface have formed the opening that comprises nanoporous.
14. a method that is used for the characterising biological molecule may further comprise the steps:
The nanohole array of the electrically addressable that comprises insulating material part is provided, wherein, described insulating material part is designed and arranges, so that have on its first surface along having along the groove of second direction longitudinal extension on a plurality of grooves of first direction longitudinal extension and the second surface at it, the relative first direction of described second direction forms certain angle, and, wherein, formed groove in first and second surfaces each, so that formed the opening that comprises nanoporous on the point of crossing of the groove in each groove in first surface and the second surface;
The nanohole array of described electrically addressable is positioned between the ion acceptor, and wherein at least one ion acceptor comprises the described biomolecules that will characterize; With
Allow described biomolecules from a nanoporous of the nanohole array of the electrically addressable that provided, pass through.
15., further comprising the steps of as the method for claim 14:
When described biomolecules is passed through one of described nanoporous, detect the change of ion(ic)current and ion(ic)current; With
According to the change of detected ion(ic)current and ion(ic)current, characterize described biomolecules.
16. a method that is used for determining the RNA secondary structure may further comprise the steps:
The nanohole array of the electrically addressable that comprises insulating material part is provided, wherein, described insulating material part is designed and arranges, so that have on its first surface along having along the groove of second direction longitudinal extension on a plurality of grooves of first direction longitudinal extension and the second surface at it, the relative first direction of described second direction forms certain angle, and, wherein, described groove is to form in first and second surfaces each, so that formed the opening that comprises nanoporous on the point of crossing of the groove in each groove in first surface and the second surface;
The nanohole array of described electrically addressable is positioned between the ion acceptor, and wherein at least one ion acceptor comprises the described biomolecules that will characterize;
Functionally the end with RNA is coupled on the optical tweezers; With
When being pulled through one of described nanoporous, measures described RNA molecule the power of a described end.
17. a nanoporous comprises:
Insulating element with apparent surface; With
The through hole that between described apparent surface, extends.
18. as the nanoporous of claim 17, wherein, the width of described through hole is less than about 10nm.
19. a nanohole array that comprises a plurality of nanoporouss, wherein, each nanoporous comprises:
Insulating element with apparent surface; With
The through hole that between described apparent surface, extends.
20. as the nanohole array of claim 19, wherein, the width of described through hole is less than about 10nm.
21. as the nanohole array of claim 19, wherein, each in the described nanoporous is an electrically addressable.
22. a method for preparing nanoporous, described nanoporous comprise insulating element with apparent surface and the through hole that extends between described surface, this method may further comprise the steps:
Formation has the hole of the width of determining by fixing a plurality of crystal, and described crystal is fixed with certain position each other;
By insulating material being cast in each other to form insulating element on the described a plurality of crystal of certain position fixed, so that form described insulating material, it has the described hole of extending between the apparent surface of described insulating material; With
Remove described a plurality of crystal,
So that preparation nanoporous, this nanoporous comprise insulating element with apparent surface and the through hole that extends between described surface.
23. comprise the nanoporous of insulating element with apparent surface and the through hole that extends between described surface, described nanoporous is to prepare by the method that may further comprise the steps:
Formation has the hole of the width of determining by fixing a plurality of crystal, and described crystal is fixed with certain position each other;
By insulating material being cast in each other to form insulating element on the described a plurality of crystal of certain position fixed, so that form described insulating material, it has the described hole of extending between the apparent surface of described insulating material; With
Remove described a plurality of crystal,
So that preparation comprises the nanoporous of insulating element with apparent surface and the through hole that extends between described surface.
24. prepare the method for nanohole array, described nanohole array comprises the insulating element with first and second relative surfaces, and the one or more through holes that extend between described surface, this method may further comprise the steps:
On first surface, form one or more grooves along the first direction longitudinal extension;
Form the one or more grooves along the first direction longitudinal extension on second surface, described second direction and first direction form certain angle;
Remove the material on the point of crossing of the one or more grooves that are positioned on the first surface and the one or more grooves on the second surface;
So that preparation comprises the nanoporous of insulating element with apparent surface and the one or more through holes that extend between described surface.
25. device as claimed in claim 2, wherein, described crystalline limit is (Se) in a certain distance apart from one another, and sets crystal apart from spacing, so that satisfy (Se/Tm)≤0.5, wherein, Tm is a molecular thickness of forming the material of described insulating element.
26. device as claimed in claim 2, wherein, described crystalline limit is (Se) in a certain distance apart from one another, and sets crystal apart from spacing, so that make in its scope that is in about 1 -about 10 .
27. device as claimed in claim 1, wherein, the length of described through hole is less than or equal to 20 .
28. device as claimed in claim 1, wherein, the length of described through hole (d) satisfies following relational expression: 2 ≤d≤10 .
29. method as claimed in claim 5, wherein, the length of the described through hole of the insulating element that is provided is less than or equal to 20 .
30. method as claimed in claim 1, wherein, the length of the described through hole of the insulating element that is provided (d) satisfies following relational expression: 2 ≤d≤10 .
31. method as claimed in claim 5, wherein, described crystalline limit is (Se) in a certain distance apart from one another, and sets crystal apart from spacing, so that satisfy (Se/Tm)≤0.5, wherein, Tm is a molecular thickness of forming the material of described insulating element.
32. method as claimed in claim 5, wherein, described crystalline limit is (Se) in a certain distance apart from one another, and sets crystal apart from spacing, so that make in its scope that is in about 1 -about 10 .
33. method as claimed in claim 7, wherein, the length of the described through hole of the insulating element that is provided is less than or equal to 20 .
34. method as claimed in claim 7, wherein, the length of the described through hole of the insulating element that is provided (d) satisfies following relational expression 2 ≤d≤10 .
35. method as claimed in claim 7, wherein, described crystalline limit is (Se) in a certain distance apart from one another, and sets crystal apart from spacing, so that satisfy (Se/Tm)≤0.5 wherein, Tm is a molecular thickness of forming the material of described insulating element.
36. method as claimed in claim 7, wherein, described crystalline limit is (Se) in a certain distance apart from one another, and sets crystal apart from spacing, so that make in its scope that is in about 1 -about 10 .
37. a method for preparing the two-dimensional nano hole array that comprises insulating element with apparent surface and a plurality of through holes that extend between described surface, this method may further comprise the steps:
Form each in described a plurality of hole, the width in each hole determines that by fixing a plurality of crystal described crystal is fixed with certain position each other;
Form insulating element by insulating material being cast in each other on each for each hole with the described a plurality of crystalline of certain position fixed, so that form described insulating material, it has described each hole of extending between the apparent surface of described insulating material; With
Remove described a plurality of crystal in each hole,
So that preparation comprises the two-dimensional nano hole array of insulating element with apparent surface and a plurality of through holes that extend between described surface.
38. prepare the method for nanohole array, may further comprise the steps:
A plurality of nanoporous assemblies are provided, and each in described a plurality of nanoporous assemblies comprises insulating element with apparent surface and the through hole that extends between described surface,
Assemble a plurality of nanoporous assemblies, so that described through hole has formed the one-dimensional array at least of through hole; With
Wherein, describedly provide a plurality of nanoporous assemblies to comprise each nanoporous assembly is implemented following steps:
Form the hole, its width determines that by fixing a plurality of crystal described crystal is fixed with certain position each other,
By insulating material being cast in each other to form insulating element on the described a plurality of crystal of certain position fixed so that form insulating material, its have the described hole of between the apparent surface of described insulating material, extending and
Remove described a plurality of crystal, so that preparation comprises the nanoporous of insulating element with apparent surface and the through hole that extends between described surface.
39. as the method for claim 38, wherein, a plurality of nanoporous assemblies of described assembling comprise a plurality of nanoporous assemblies of assembling, so that described through hole forms the two-dimensional array of through hole.
40. as method any among the claim 38-39, wherein, the length of the described through hole of each of a plurality of nanoporous assemblies that provide is less than or equal to 20 .
41. as method any among the claim 38-39, wherein, the length (d) of the described through hole of each of a plurality of nanoporous assemblies that provide satisfies following relational expression: 2 ≤d≤10 .
42. as method any among the claim 38-39, wherein, described formation hole comprises makes crystalline limit (Se) in a certain distance apart from one another, wherein set described distance, so that satisfy following relation (Se/Tm)≤0.5, wherein, Tm is a molecular thickness of forming the material of described insulating element.
43. as method any among the claim 38-39, wherein, described formation hole comprises makes crystalline limit (Se) in a certain distance apart from one another, wherein sets described distance, so that make in its scope that is in about 1 -about 10 .
44. the method to DNA or RNA check order may further comprise the steps:
Insulating element is provided, and described insulating element is included in the through hole that extends between the apparent surface, and wherein, the width of described through hole is to be determined by a plurality of crystal of arranging relatively with fixed relationship each other when forming insulating element;
At least the internal surface of through hole is applied with PNA, described PNA has comprised the A that characterizes DNA/RNA, T, a kind of in G or the C base;
Described insulating element is positioned between the ion acceptor, and wherein at least one ion acceptor comprises the DNA or the RNA that will check order;
Functionally the end with DNA/RNA is coupled on the optical tweezers;
When being pulled through described through hole, described DNA/RNA measures power as the function of time at a described end; With
Make power and A as the described measurement of the function of time, T, a kind of being correlated with in G or the C base checked order to DNA or RNA so that prepare.
45. the method to DNA or RNA check order may further comprise the steps:
A plurality of nanoporous assemblies are provided, and each in described a plurality of nanoporous assemblies comprises insulating element with apparent surface and the through hole that extends between described surface,
Assemble described a plurality of nanoporous assembly, so that described through hole has formed the one-dimensional array of through hole at least;
With PNA each the internal surface at least of through hole in described a plurality of nanoporous assemblies is applied, described PNA has comprised the A that characterizes DNA/RNA, T, a kind of in G or the C base;
Described insulating element to each nanoporous assembly positions, and is deployed at least one side of described a plurality of nanoporous assemblies so that will comprise the ion acceptor of the DNA/RNA that will check order;
Functionally the end with the DNA/RNA of the through hole of described at least one nanoporous assembly is coupled on the optical tweezers;
When being pulled through the through hole of described at least one nanoporous assembly, described DNA/RNA measures power as the function of time at a described end;
Will be as the power and the A of the described measurement of the function of time, T, a kind of relevant in G or the C base is so that check order to described DNA or RNA; With
Wherein, describedly provide a plurality of nanoporous assemblies to comprise each nanoporous assembly is implemented following steps:
Formation has the hole of the width of determining by fixing a plurality of crystal, and described crystal is fixed with certain position each other,
By insulating material being cast in each other to form insulating element on the described a plurality of crystal of certain position fixed, so that form described insulating material, it has the described hole that the apparent surface by described insulating material extends,
Remove described a plurality of crystal, and
At least the internal surface of described through hole is applied with PNA, described PNA has comprised the A that characterizes DNA/RNA, T, a kind of in G or the C base.
46. device as claimed in claim 1 also comprises the PNA coating, described PNA comprises A, T, and a kind of in G or the C base, described base characterizes DNA/RNA; Wherein, described coating is coated on the internal surface of described through hole at least.
47. the nanohole array of electrically addressable as claimed in claim 8 also comprises the PNA coating, described PNA comprises A, T, and a kind of in G or the C base, described base characterizes DNA/RNA; Wherein, described coating is coated on the internal surface of opening at least, and these openings are to form on the point of crossing of described first and second lip-deep each groove.
48. as the nanoporous of claim 17, also comprise the PNA coating, described PNA comprises A, T, and a kind of in G or the C base, described base characterizes DNA/RNA; Wherein, described coating is coated on the internal surface of described through hole at least.
49. as the nanohole array of claim 19, also comprise the PNA coating, described PNA comprises A, T, and a kind of in G or the C base, described base characterizes DNA/RNA; Wherein, described coating is coated on each the internal surface of through hole of described a plurality of nanoporouss at least.
50. the method to DNA or RNA check order may further comprise the steps:
Provide device, as the nanohole array of the electrically addressable of claim 47, as the nanoporous of claim 48 with as in the nanohole array of claim 49 any one as claim 46;
With described device, the nanohole array of described electrically addressable, any one in described nanoporous and the described nanohole array is positioned between the ion acceptor, and wherein at least one ion acceptor comprises the DNA or the RNA that will check order;
Functionally the end with DNA/RNA is coupled on the optical tweezers;
When being pulled through described through hole, described DNA/RNA measures power as the function of time at a described end; With
Will be as the power and the A of the described measurement of the function of time, T, a kind of relevant in G or the C base,
So that prepare DNA or RNA are checked order.
51. as the method that is used to prepare nanoporous of claim 22, further comprising the steps of: at least the internal surface of described through hole is applied with PNA, described PNA has comprised the A that characterizes DNA/RNA, T, a kind of in G or the C base.
52. as the method that is used to prepare nanohole array of claim 24, further comprising the steps of: at least the internal surface of one or more through holes is applied with PNA, described PNA has comprised the A that characterizes DNA/RNA, T, a kind of in G or the C base.
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