CN1997678B - Quasi-interpenetrating networks used as separation media - Google Patents

Quasi-interpenetrating networks used as separation media Download PDF

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CN1997678B
CN1997678B CN2004800270664A CN200480027066A CN1997678B CN 1997678 B CN1997678 B CN 1997678B CN 2004800270664 A CN2004800270664 A CN 2004800270664A CN 200480027066 A CN200480027066 A CN 200480027066A CN 1997678 B CN1997678 B CN 1997678B
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molecular weight
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quasi
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CN1997678A (en
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朱鹏年
宋立国
方渡飞
梁德海
刘天波
王延梅
应琦琮
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Research Foundation of State University of New York
New York University NYU
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L33/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Compositions of derivatives of such polymers
    • C08L33/24—Homopolymers or copolymers of amides or imides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2205/00—Polymer mixtures characterised by other features
    • C08L2205/02—Polymer mixtures characterised by other features containing two or more polymers of the same C08L -group
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2205/00—Polymer mixtures characterised by other features
    • C08L2205/04—Polymer mixtures characterised by other features containing interpenetrating networks

Abstract

The invention relates to a quasi-interpenetrating network of polymer chains, the chains comprising linear polyacrylamide (LPA) chains in the form of a main frame having a weight average molecular weight of approximately 0.05 million to approximately 25 million g/mole, and a radius of gyration of approximately 10 nrn to 350 rm; and polydimethylacrylamide (PDMA) chains prepared by polymerizing PDMAin the presence of the LPA main frame, wherein the LPA and PDMA chains are entangled within one another and interpenetrate one another, and wherein the quasi-interpenetrating network has substantially no chemical cross-linking.

Description

Quasi-interpenetrating networks as separating medium
The present invention finishes under the government of the national human genome fund No.5R01HG01386-07-8 of EASD supports.Government enjoys some right of the present invention.
Technical field
The present invention relates in general to the technical field of separating charged minute subclass material, relates in particular to the separating medium that is used for capillary electrophoresis.
Background technology
In the biology related science, gel electrophoresis is a kind of the most widely used isolation technique.Under the effect of extra electric field, by making charged molecule class material, as protein, peptide class, nucleic acid and oligonucleotide, migration takes place in buffer medium they are separated.Usually, buffer medium is used with a kind of low gelifying agent that is fit to intermediate concentration, separate with promotion, and separated material generation blended probability is reduced to minimum.
Up to date, electrophoretic separation is just carried out on gel slab or open gel bed, and it normally is made of agarose or crosslinked polyacrylamide material.Recently, use polymer gel or solution to be used to the separation of charged molecule such as DNA as the capillary electrophoresis (CE) of separating medium.The unite automated operation and the fast quantitative analysis that use to charged molecule of capillary electrophoresis technique and photometric detection method provide possibility.And with respect to traditional disk electrophoresis method, capillary electrophoresis can be at the quantitative information that uses sampling under the situation of very a spot of sample, gel (or polymers soln) and buffer reagent.And, realized having the charged macromolecular high-resolution separation of different effective charges.
Usually, the used capillary column of capillary electrophoresis is prepared by fused quartz tube, its diameter greatly about 25 μ m between the 200 μ m, length is about 30cm to about 200cm.Gel or solution separating medium and buffer reagent have been full of in the capillary column.Electrophoretic technique is used to separating charged minute subclass material.
In CE, separating medium is one of most important parameter, because the migration feature of its decision charged molecule class material (as dna fragmentation) comprises resolving power.At present, polymers soln is widely used in such separating medium.
Developed the hydrophilic homopolymer of several synthetic, verified as the DNA separating medium simultaneously.These homopolymer comprise: linear polyacrylamide (LPA); And alkyl-substituted derivatives; as poly--N, N--DMAA (PDMA), poly--N-acryl amino-ethoxy ethanol (PAAEE), polypropylene acyl group aminopropanol (PAAP), poly-(acryl amino ethoxy) ethyl glycopyranoside (PAEG), polyoxyethylene glycol (PEG) and polyethylene oxide (PEO).
Electrophoresis circle is admitted very much: highly the LPA solution of Chan Jieing can provide best order-checking (sequencing) to separate usually, can separate in the fragment that reaches 1300 bases and have only a dna molecular (Zhou et al., Anal.Chem.72: 1045-52 (2000)) that base is different.Think that the good performance of LPA partly is because its wetting ability.
Although LPA can provide good DNA to separate for long read to check order (long read sequencing), it has requirement to use the shortcoming of stablizing the capillary wall coating.This capillary wall coating requires can suppress electroosmotic flow (electro-osmotic flow) (EOF), and prevents the absorption of assay.The generation of electroosmotic flow is because separating medium can not directly connect with capillary tube inner wall.Apply electric field in sepn process, the electroosmotic flow that this problem is serious will occur.Can form electrostatic double layer by the powered surfaces that solvable buffer ions is adsorbed onto capillary wall.In near the capillary wall the solution, the ion partial concn is excessive, and this will cause keeping neutral charge at the position of adjacent capillaries wall.Net result is: in electrophoresis process, can form the overall flow of liquid at charged conduit wall.Electroosmotic flow causes the separating resulting that makes us being not satisfied at all.
Be intended to prevent that the traditional method that EOF produces from comprising the importing compound, before separating medium was injected kapillary, this compound can be incorporated on the internal surface of capillary wall.As the U.S. Patent No. 5,447,617 of authorizing Shieh described polyhutadiene has been covalently bound on the internal surface capillaceous, introduces acrylamide monomer therein, and with acrylamide and polyhutadiene copolymerization.This precoated layer technology not only increases cost, also produces following problem: as kapillary incrustation, coating heterogeneity and applied capillaceous limited storage life.
For avoiding the generation of EOF problem, the polymkeric substance that wetting ability is lower is used as separating medium and uses.Such as: poly-(vinyl pyrrolidone) (PVP) uses as dna sequencing matrix.Because PVP can dynamically be coated with capillary wall, has avoided the demand for the precoating capillary wall.But, compare the separation efficiency difference of PVP many with the separation efficiency that uses LPA to obtain.Use PVP only can realize being slightly larger than the separation of 300 continuous DNA bases.The restricted of PVP is characterised in that: screening matrix and non-specific (hydrophobicity) between the fluorescence dye used between separation period interact too high.This interaction makes the segmental separation of larger dna unclear.
The exploitation of high-performance separating medium (its screening capacity height, viscosity are low, and have dynamic coating ability) will help the automatization of CE, further improve its performance.But the homopolymer with these characteristics is not also found up to now.Therefore, the mixture of different homopolymer is studied.
Discover: (as PEO, Hexaethyl Mierocrystalline cellulose (HEC) and mixture LPA), and the mixture of two kinds of modified polysaccharide as agarose and HEC, all have higher resolving power for DNA small segment and the big fragment of DNA to the different same polymkeric substance of molecular weight.But, by the diverse two kinds of mixtures that mixed with polymers obtains of chemical structure, the application of never succeeding.People such as Kim (Kim, Y., Yeung, E.S., J.Chromatogr.A., 1997,781,315-325) once attempted using the mixture of PEO and six propyl celluloses (HPC) to carry out dna sequencing, but found the non-constant of separating effect.The reason of failure is the uncompatibility of two kinds of polymkeric substance.
The another one challenge of CE is: common very heavy-gravity isolation medium is imported in the narrow kapillary.Polyacrylamide is polymerization in kapillary, has avoided forcing polymers soln to enter the problem of kapillary or microchannel.Also wish after each the use, " used " polymeric matrix to be removed from kapillary, and in kapillary, fill fresh matrix again.But in case polyacrylamide polymerization in kapillary, the gel after the polymerization is difficult to remove from kapillary.
Recently, having the multipolymer that viscosity relies on feature is employed.This multipolymer allows the loading and the removal of medium under the low viscosity state, and the electrophoretic separation under the high viscosity state.As: the matrix based on the hydrophilic skeleton of the LPA of the grafts of the weak point with lower mol (N-N-isopropylacrylamide) is described (Sudor et al.Electrophoresis22: 720-8 (2001)).And, based on N,N-DMAA and N, the matrix also detected (Buchholzet al.Anal Chem 73: 157-64. (2001)) of the multipolymer of N-diethyl acrylamide (" P (DMA/DEA) ").But synthetic being difficult to of multipolymer controlled and repeats, and can produce insecure result.
Successful electrophoresis will comprise following feature with separating medium: stable, neutral, suitable mesh size, dynamically be coated with ability (with effective inhibition electric osmose), low viscosity, the dielectric material consumption is minimum, good separating resulting can be provided simultaneously and long read long (read length).Before the present invention, the medium of some desired characteristic (as the long length of reading) can be provided, generally do not possess other desired characteristic simultaneously, as dynamic coating ability.
Summary of the invention
In one embodiment, the invention provides a kind of quasi-interpenetrating networks (class-IPN) of polymer chain, this polymer chain comprises: linear polyacrylamide (LPA) chain that exists with main skeleton form, and polydimethylacrylamiin (PDMA) chain, the weight-average molecular weight of LPA chain is about 0.05 * 10 6To about 25 * 10 6G/mol, rotation radius approximately is that 10nm is to 350nm; And polydimethylacrylamiin (PDMA) chain is when main skeleton LPA exists, and prepares by polymerization PDMA.Wherein, LPA chain and PDMA chain tangle each other, IPN; And wherein, such-IPN do not have chemically crosslinked substantially.
In another specific embodiment, the invention provides the quasi-interpenetrating networks of the polymer chain of entanglement, this polymer chain comprises linear polyacrylamide (LPA) chain and polydimethylacrylamiin (PDMA) chain, this PDMA chain entanglement is at the LPA interchain, and with LPA chain IPN, wherein the weight-average molecular weight of LPA and PDMA approximately is 0.1 * 10 6To about 20 * 10 6G/mol, and rotation radius approximately be 15nm to 320nm, wherein such-IPN do not have chemically crosslinked substantially.
In another specific embodiment, the invention provides the quasi-interpenetrating networks of the polymer chain of entanglement, it is prepared by following method.This method comprises: the solution that comprises linear polyacrylamide (LPA) and buffer reagent is provided, and wherein this LPA weight-average molecular weight is 0.05 * 10 6To 25 * 10 6G/mol; The solution that comprises polydimethylacrylamiin (PDMA) and buffer reagent is provided, and wherein the weight-average molecular weight of this PDMA is 100,000 to 25 * 10 6G/mol; Mix with the solution of PDMA/ buffer reagent with the solution of substep mode the LPA/ buffer reagent, wherein the concentration of LPA/ buffer agent solution is 1 to 15 times of PDMA/ buffer agent solution concentration, and the volume of LPA/ buffer agent solution approximately is 1 to 50 times of PDMA/ buffer agent solution volume; Wherein, prepare the LPA of entanglement, IPN and the quasi-interpenetrating networks of PDMA polymer chain, wherein, class-IPN does not have chemically crosslinked substantially.
In another specific embodiment, the invention provides the method for separating charged minute subclass material.This method is included under the influence of extra electric field, and charged molecule class material is moved in separating medium, and wherein the improvement of separating medium comprises: LPA polymeric system and PMDA polymeric system, wherein this polymer system forms quasi-interpenetrating networks.
In another specific embodiment, the invention provides a kind of quasi-interpenetrating networks of polymer chain, this polymer chain comprises acrylamide (AM)/DMAA (DMA) the random copolymers chain that exists with main skeleton form, and its weight-average molecular weight is about 0.05 * 10 6To about 2 * 10 6G/mol, rotation radius is that about 10nm arrives about 80nm, polydimethylacrylamiin (PDMA) chain is that polymerization PDMA prepares by in the presence of the main skeleton of this random copolymers; Wherein, described chain tangles each other, IPN, and wherein, such-IPN do not have chemically crosslinked substantially.
In another specific embodiment, the invention provides a kind of quasi-interpenetrating networks that constitutes by the polymer chain that tangles, it is prepared by following method, this method comprises: the solution of a kind of AM/DMA of comprising random copolymers and buffer reagent is provided, and wherein the weight-average molecular weight of AM/DMA random copolymers is 0.05 * 10 6To 2 * 10 6G/mol; A kind of solution that comprises polydimethylacrylamiin (PDMA) and buffer reagent is provided, and wherein the weight-average molecular weight of PDMA is 0.05 * 10 6To 25 * 10 6G/mol; In the substep mode this multipolymer/buffer agent solution and PDMA/ buffer agent solution are mixed, wherein the concentration of this multipolymer/buffer agent solution is 1 to 50 times of PDMA/ buffer agent solution concentration, and the volume of this multipolymer/buffer agent solution is about 1 to 20 times of PDMA/ buffer agent solution volume; Wherein, prepare the multipolymer of entanglement and the quasi-interpenetrating networks of PDMA polymer chain.
In another specific embodiment, the invention provides the method for a kind of separating charged minute subclass material.This method comprises: under the influence of extra electric field, charged molecule class material is moved in separating medium, wherein the improvement of this separating medium comprises: AM/DMA random copolymers and PMDA polymkeric substance, wherein, this polymeric system forms quasi-interpenetrating networks.
Description of drawings
Fig. 1 is the Zimm plot of sample LPA in 1 * TTE buffered soln.The figure shows in scattering angle 15 0To 139 0, when temperature was 25 ℃, the static light scattering of LPA was measured.
Fig. 2 is the Zimm plot of sample quasi-interpenetrating networks in 1 * TTE buffered soln.The figure shows in scattering angle is 15 0To 139 0, when temperature is 25 ℃, comprise that the static light scattering of the quasi-interpenetrating networks of LPA and PDMA is measured.
The apparent hydrodynamic radius that Fig. 3 shows sample LPA distributes, and it is 30 in scattering angle 0, temperature is 25 ℃, C=3.387 * 10 -5Obtain by dynamic light scattering measurement during g/ml.
Fig. 4 shows the apparent hydrodynamic radius of sample quasi-interpenetrating networks and distributes, and it is to be 30 in scattering angle 0, temperature is 25 ℃, C=3.201 * 10 -5Obtain by dynamic light scattering measurement during g/ml.
Fig. 5 shows by CE DNA isolation (pGEM-3Zf (+) from-21M13 upstream primer).This is separated in IPN/1 * TTE+7M urea buffered soln, and C=2.0%g/ml carries out under 60 ℃ of conditions.Useful length capillaceous is 34cm, ID/OD=75/365 (μ m).Be separated in this laboratory self-made Instrument and carry out.
Fig. 6 shows by CE DNA isolation (pGEM-3Zf (+) from-21M13 upstream primer).This is separated in MegaBACE LPA/l * MegaBACE damping fluid, 44 ℃, carries out under the condition of 150V/cm.Length capillaceous is 34cm, ID/OD=75/365 (μ m).Be separated in this laboratory self-made Instrument and carry out.
Fig. 7 shows by CE DNA isolation (pGEM-3Zf (+) from-21M13 upstream primer).This is separated in POP6/1 * TTE damping fluid, 50 ℃, carries out under the condition of 200V/cm.Length capillaceous is 34cm, ID/OD=75/365 (μ m).Be separated in this laboratory self-made Instrument and carry out.
Fig. 8 shows the terminal portions (base number from 600 to～1000) by CE electrophoretic separation DNA BigDye Terminator v3.0.This is separated in quasi-interpenetrating networks/1 * TTE+7M urea buffered soln, and C=2.0%g/ml carries out under 60 ℃ the condition.Instrument: ABI Prism 310 4 look single capillary genetic analyzers.
Fig. 9 shows the blueness-G part by CE electrophoretic separation DNA (BigDye Terminator v3.0).This is separated in POP6/1 * ABI buffered soln and carries out.Instrument: ABI Prism 310 4 look single capillary genetic analyzers, 50 ℃, Module seqPOP6 (1ml) E.Kapillary ID/OD=50/361 (μ m).
Figure 10 shows by using laboratory self-control sequencing analysis instrument, with 2.5% class-IPN/1 * ABI damping fluid as separating medium, four look DNA (the BigDye Terminator v3.0) sequence of the 660bp that is being less than～is recording in the 33min.(instrument and base calling software are provided by the Engineering College of Stony Brook university)
Figure 11 shows by using laboratory self-control sequencing analysis instrument, with POP7/1 * ABI damping fluid as separating medium, the four look dna sequence dnas (BigDye Terminator v3.0) of 600 bases that in～63min, record.(instrument and base calling software are provided by the Engineering College of StonyBrook university)
Figure 12 shows the separation of DNA in LPA/PDMA quasi-interpenetrating networks medium (BigDyeTerminator v3.0), wherein LPA (weighted average MW=7.6 * 10 6G/mol) and PDMA (content of weighted average MW～470Kg/mol) is respectively 83% and 17%.Operating voltage 195.5V/cm, the DNA injection length is 20s under 27V/cm.The kapillary useful length is 33cm, ID/OD=50/361 (μ m).Instrument: ABI Prism 310 4 look single capillary genetic analyzers.
Figure 13 shows the separation of DNA (BigDyeTerminator v3.0) in quasi-interpenetrating networks (20805) medium.In the quasi-interpenetrating networks content of PDMA and LPA be respectively～11% and～89%.Operating voltage 195.5V/cm, the DNA injection length is 20s under 27V/cm.The kapillary useful length is 33cm, ID/OD=50/361 (μ m).Instrument: ABIPrism 310 4 look single capillary genetic analyzers.
Figure 14 shows and changes the DNA injecting voltage: (a) 25V/cm; (b) 75V/cm; (c) 100V/cm is to the influence of the isolating resolving power of DNA.This is separated in the self-made Instrument of laboratory and carries out.Injection length is 10 seconds; The kapillary useful length is 35cm; Kapillary ID=75um; Applied electric field=150V/cm; Buffered soln is 1 * TTE/7M urea; DNA:pGEM3Zf (+) (C-mix).
Figure 15 shows at effective column length and is (a) 35cm and (b) variation of DNA isolation resolution during 40cm.Be separated in the self-made Instrument of laboratory and carry out, injecting voltage and time are 75V/cm, 10 seconds.Instrument working conditions and DNA sample are with described in Figure 14.
Figure 16 shows applied electric field intensity and is (a) 200V/cm and (b) electrophorogram of the 581st base back of pGEM3Zf (+) during 150V/cm.Be separated in the self-made Instrument of laboratory and carry out, injecting voltage and time are 75V/cm, 10 seconds.Kapillary useful length=40cm.Instrument working conditions and DNA sample are with described in Figure 14.
Figure 17 shows by using by LPA and PDMA ratio and is (a) 1: 0 (uncoated kapillary); (b) 1.62: 1; (c) 3.33: 1; (d) quasi-interpenetrating networks of 6.33: 1 (e) 10.9:1 is as the separation of the dna sequencing sample of separating medium.Be separated in the self-made Instrument of laboratory and carry out, injecting voltage and time are 75V/cm, 10 seconds.Kapillary useful length=40cm.Instrument working conditions and DNA sample are with described in Figure 14.
Figure 18 shows different LPA molecular weight: (a) Mv=4.53 * 10 6G/mol; (b) Mv=6.4 * 10 6G/mol; (c) Mv=7.69 * 10 6G/mol; (d) Mv=9.91 * 10 6G/mol is to the isolating influence of DNA.Be separated in the self-made Instrument of laboratory and carry out, injecting voltage and time are 75V/cm, 10 seconds.Kapillary useful length=40cm.Instrument working conditions and DNA sample are with described in Figure 14.
Figure 19 shows the comparison of DNA separating resulting, and wherein LPA is by the polymerization of (a) emulsifier-free; (b) conversed phase micro emulsion copolymerization prepares.Be separated in the self-made Instrument of laboratory and carry out, injecting voltage and time are 75V/cm, 10 seconds.Kapillary useful length=40cm.Instrument working conditions and DNA sample are with described in Figure 14.
Figure 20 shows the yellow-T part of the electrophorogram of DNA (BigDye Terminator v3.0).Instrument and working conditions are with identical among Fig. 8.
Figure 21 shows the single base resolving power in DNA (BigDye Terminator v3.0) electrophorogram yellow-T part, as shown in figure 20.
Embodiment
The present invention relates to be used for separating charged macromolecular capillary electrophoresis separating medium, and the method for preparing and use this separating medium.
Separating medium among the present invention comprises " quasi-interpenetrating networks " (class-IPN) of a kind of polymkeric substance or copolymerization chain.These chains are IPN, entangled to each other each other.In a specific embodiment, class-IPN comprises linear polyacrylamide (LPA) chain and polydimethylacrylamiin (PDMA) chain.In another specific embodiment, class-IPN comprises the random copolymers that is made of acrylamide (AM) and DMAA (DMA) chain, and the polydimethylacrylamiin chain.
Chemically crosslinked does not require and appears among class-IPN.Preferably, chemically crosslinked does not appear.But, because a small amount of chemically crosslinked may appear in the experiment condition difference.Based on the purpose of this specification sheets, the definition of a small amount of chemically crosslinked is: be lower than about 5%, 2%, 1%, 0.5% or 0.2% polymer chain and other polymer chain generation chemically crosslinked.In the preparation of class-IPN, do not use linking agent.
Generally speaking, in the art, term " interpenetrating(polymer)networks " is used to describe a kind of paradigmatic structure, and this structure comprises at least two kinds of independently chemically crosslinked polymer networks, and this network is IPN each other, but does not take place crosslinked mutually.Therefore, the difference that exists between known interpenetrating(polymer)networks of prior art and quasi-interpenetrating networks involved in the present invention is: this quasi-interpenetrating networks has been formed a kind of single network of not having chemically crosslinked substantially.
Such-every kind of polymkeric substance (or multipolymer) among the IPN provides for the different desired characteristic of separating medium.As the LPA possess hydrophilic property, and provide good separation efficiency, as the resolving power height, read long long and the working hour short.But because LPA can not directly be connected with capillary tube inner wall, this causes producing undesirable electroosmotic flow in electrophoresis process.
The PDMA wetting ability is relatively poor, can dynamically be coated with capillary tube inner wall, therefore can avoid the generation of electroosmotic flow.Simultaneously, PDMA can suppress the non-specific adsorption of capillary wall to charged macromole (as dna fragmentation).
The present invention passes through these two kinds of different polymkeric substance, and LPA and PDMA combine in class-IPN, and every kind of polymkeric substance Ideal Characteristics is attached in a kind of separating medium.
Generally speaking, in solvent, the consistency of LPA and PDMA chain is from miscible to not miscible mutually each other each other.Based on the purpose of this specification sheets, if LPA and the PDMA chain can mix and can keep dissolved state over a long time then is defined as LPA and PDMA chain miscible; Even if when the uniform solution that the height that obtains LPA and PDMA chain tangles, LPA and PDMA chain finally also will be separated, and then LPA and PDMA chain are defined as unmixing.If LPA and PDMA chain have the compatibility to a certain degree between miscible and immiscible polymer chain, then LPA and PDMA chain are the part unmixings.
The compatibility of LPA and PDMA chain is subjected to the influence of Several Factors, as the molecular weight of total polymer concentration, polymkeric substance in the solvent and the relative content of every kind of polymkeric substance.Along with the increase of total polymer concentration, and/or the polymericular weight increase, and/or LPA increases the compatibility reduction as PDMA relative content in the solution of main ingredient.
Obtain to be used for the suitable mesh size of separating medium, require to have certain minimum total polymer concentration.Generally speaking, be to obtain that order-checking separates the mesh size that is fit to for the overwhelming majority, requiring total polymer concentration approximately is the order of magnitude of 1.5wt% or higher.Suitable with PDMA chain relative content at LPA, when its molecular weight was high simultaneously, when total polymer concentration was 1.5wt%, LPA and PDMA chain be the part unmixing each other.But by making the immiscible LPA chain of part and PDMA chain formation class-IPN, in case when needs carry out the separation of charged biomolecules, this LPA chain and PDMA chain should be able to tangle each other, IPN at least.
LPA chain in the quasi-interpenetrating networks and PDMA chain are inequality on polymer chain repels each other this aspect at least.Therefore, the stretching, extension that becomes in solution of this chain surpasses its normal conformation.Therefore, compare with the composing type LPA chain that does not exist with the quasi-interpenetrating networks form and the by weight/volume sum of PDMA chain, the by weight/volume of the quasi-interpenetrating networks in the solution is littler.More wide-spread conformational energy effectively provides a kind of better separating medium, and the quantity that will form the required polymer materials of ideal mesh size simultaneously reduces to minimum degree.
LPA and PDMA chain must be miscible to such degree, promptly at least in case when needs carry out charged macromolecular separation, this polymer chain should be able to keep tangling each other.So, when quasi-interpenetrating networks was placed in the order-checking buffer medium, this quasi-interpenetrating networks existed with solution form homogeneous, gelatinous.
Some physical property of LPA chain, PDMA chain and quasi-interpenetrating networks can be determined.These characteristics comprise molecular weight, polydispersity index, rotation radius and hydrodynamic radius.The separation efficiency that is provided by specific quasi-interpenetrating networks can be provided these characteristics.Based on the concrete application of quasi-interpenetrating networks, these characteristics can be changed with the optimization separating resulting.These characteristics will be inquired into hereinafter.
The molecular weight of single polymers chain obtains by amount of monomer in monomeric molecular weight and the polymer chain is simply multiplied each other.Comprise multiple polymer chain in the polymers soln with different molecular weight.The molecular weight of polymkeric substance promptly is the mean value of the molecular weight of polymer chain in the solution in the solution.There are several modes to describe the molecular-weight average of polymkeric substance in the solution.
A kind of mode of polymericular weight in the solution of describing is by determining polymkeric substance " number-average molecular weight " (M in the solution n).Number-average molecular weight is defined as the total quantity of the total molecular weight of polymer chain in the solution divided by polymer chain in the solution.That is to say Mn=∑ niMi/ ∑ n i, n wherein iBe that molecular weight is M in the solution iThe number of polymer chain.
The another kind of mode of describing polymericular weight is by determining " weight-average molecular weight " (M w).Weight-average molecular weight is defined by n iM i 2Summation divided by n iM iSummation, n wherein jBe that molecular weight is M iThe number of polymer chain.That is to say M w∑ n iM i 2/ ∑ n iM iWeight-average molecular weight can be measured by static Experiment of Laser Scattering.
Ratio M w/ M nBe defined by the polydispersity index of polymkeric substance.This index is that the distribution of sizes of polymer chain is measured.The weight-average molecular weight of polydispersion material is generally big than number-average molecular weight.If the value of polydispersity index is near 1, then the polymer chain size is more approaching.As well known by the skilled person in the art, polydispersity index can use numerical method, i.e. CONTIN method (Provencher, Comput.Phys.Commun.27: 229 (1982)) and in conjunction with the estimation of dynamic light scattering assay method obtain.
Rotation radius (R g) be the size measures of polymer chain.It is to be m according to molecular weight in the polymer chain iEach monomer define i.e.: R apart from the range distribution of polymer chain center of gravity g 2=∑ m ir i 2The rotation radius of polymkeric substance can be tested definite by static Experiment of Laser Scattering in the solution.Rotation radius is measured after overlapping concentration.
In addition, if the rotation radius R of specific polymkeric substance in the known solution gWith corresponding weight-average molecular weight (M w) two values, in same solution, have different known M so wPolymkeric substance, its rotation radius can be estimated and obtains.Formula: R below this evaluation method uses g=K gM w α, wherein, K gIt is constant.In a kind of good solvent of admitting for those skilled in the art, index α approximately is 0.58.
The characteristic that rotation radius is relevant is hydrodynamic radius (R h).By using dynamic light scattering experiment to measure the mobility (according to the translation spread coefficient in the dilute solution) of polymer molecule, can determine the hydrodynamic radius of polymer molecule in the solution.Just as known to those skilled in the art, R g: R hRatio depends on the conformation of polymer molecule.
Quasi-interpenetrating networks among the present invention can not be by being mixed with LPA and PDMA simply, and be to use specific synthetic method, with entanglement and the IPN that influences these polymer chains.
A kind of synthetic method that preferably is used to prepare quasi-interpenetrating networks comprises: by LPA, or the AM/DMA multipolymer forms polymer network, or " main skeleton ".Based on the purpose among the present invention, main skeleton comprises LPA polymer molecule or the AM/DMA copolymer molecule in the solvent, and wherein as mentioned below, for specific solvent, the concentration of solution is higher than LPA far away, or the overlapping concentration (C of AM/DMA multipolymer *).
The overlapping concentration of polymkeric substance is meant in the solution: the polymer molecule minimum concentration when tangling each other that is interweaved for the first time and becomes.Therefore, the feature of polymers soln significantly changes when overlapping concentration usually.Single polymer molecule during with dilute concentration is more compared, and the single polymer molecule during overlapping concentration is bigger to the motion effects of adjacent molecule.Such as, the motion of polymer molecule more is subject to adjacent molecule.Similarly, generally speaking, soltion viscosity sharply increases.
Overlapping concentration can decide according to the various characteristics of measuring of polymers soln.Such as, generally speaking, the overlapping concentration of polymers soln is estimated as the inverse of solution intrinsic viscosity ([η]).
And overlapping concentration can be according to the molecular weight M and the rotation radius (R of polymkeric substance in the solution g), utilize formula: C *～M/R g 3Estimation obtains.Molecular weight and rotation radius can test measuring by scattering of light known in the art.B.Chu，“Laser?Light
Scattering：Basic?Principles?and?Practice，”2 naEdition，Academic?Press，Boston(1991)pp.343。
The main skeleton of LPA forms by using any suitable polymerization process polypropylene amide monomer.In case solution reaches overlapping concentration, the main skeleton of LPA just can form.The example of polymerization process comprises: conversed phase micro emulsion copolymerization, emulsifier-free polymerization, conventional and seeded emulsion polymerization (conventional and seeded emulsion polymerization) and dispersion and suspension polymerization.
The preferred polymerization process that produces high-molecular weight LPA, as, conversed phase micro emulsion copolymerization.The conversed phase micro emulsion copolymerization method is known in the art.Reverse micro emulsion comprises the nano-scale level water droplet that is wrapped up by the oiliness molecule.At initiator, under the triggering as UV radiation or thermal initiator, polymerization takes place in water droplet.But the emulsifier-free polymerization is synthetic macromolecule amount LPA molecule also.
Preferably have range of molecular weight distributions and be rather narrow, just the little LPA of polydispersity index.Conversed phase micro emulsion copolymerization provides polydispersity index little LPA.
In whole specification sheets, have by the defined scope of upper and lower bound.Each lower limit can combine scope of definition with each upper limit.Bound all can be used as individual elements.
Measure when preferably, there is LPA in the physical properties of main skeleton LPA in buffered soln.The example of appropriate buffer reagent comprises: Tris-TAPS-EDTA (TTE), Tris-boric acid (Borate)-EDTA (TBE) and Tris-Glacial acetic acid-EDTA (TAE).The following physical properties of LPA is measured in 1 * TTE buffered soln.1 * TTE buffered soln contains 50mM Tris; 50mM TAPS; With 2.0mM EDTA.
Overlapping concentration depends on molecular weight and the solvent of LPA.The lower limit of the overlapping concentration range of LPA approximately is 5.0 * 10 among 1 * TTE -4G/ml.The example of other lower limit comprises: 5.0 * 10 -3G/ml and 9.0 * 10 -3G/ml.The upper limit of the overlapping concentration range of LPA approximately is 4.0 * 10 among 1 * TTE -2G/ml.The example of other lower limit (upper limit) comprising: 1.0 * 10 -2G/ml and 2.0 * 10 -2G/ml.The concrete example of an overlapping concentration approximately is: 1.2 * 10 -3G/ml.
Preferably, the lower limit of the weight average molecular weight range of LPA chain approximately is 0.05 * 10 6G/mol.The example of other lower limit comprises about 0.1 * 10 6, 0.3 * 10 6, 1 * 10 6, 4 * 10 6, 6 * 10 6, and 7 * 10 6G/mol.
Preferably, the upper limit of the weight average molecular weight range of LPA chain approximately is 25 * 10 6G/mol.The example of other upper limit comprises about 8 * 10 6, 10 * 10 6With 15 * 10 6G/mol.One example of LPA weight-average molecular weight is 7.6 * 10 6G/mol.
Preferably, the scope of the polydispersity index of LPA chain is narrow.Such as, preferably, the lower limit of polydispersity index scope approximately is 1.01.The example of other lower limit comprises about 1.02,1.05 and 1.1.
Preferably, the upper limit of polydispersity index scope approximately is 1.8.The example of other upper limit comprises about 1.3,1.5 and 1.6.The example of a LPA polydispersity index is 1.3.
Preferably, the lower limit of the rotation radius scope of the LPA in the buffered soln approximately is 10nm.The example of other lower limit comprises about 15nm, 28nm, 55nm, 125nm, 150nm and 165nm.
Preferably, the upper limit of the rotation radius scope of LPA approximately is 350nm in the buffered soln.The example of other upper limit comprises about 180nm, 210nm and 250nm.The example of a LPA rotation radius is about 180nm.
The R of LPA in the scope of the hydrodynamic radius of LPA and the good solvent in the solution gScope matches, and this will be known by those of skill in the art.The example of the hydrodynamic radius of LPA is about 81nm in the solution.
With DMAA monomer and the main skeleton polymerization of LPA.By adopting any suitable method polymerization DMAA monomer, as, such as, use with the identical method of described preparation LPA above.Preferred radical polymerization.
Preferably, the lower limit by the weight average molecular weight range of the quasi-interpenetrating networks of LPA and PDMA chain formation is about hundreds of thousands of.The example of other lower limit comprises about 0.1 * 10 6, 0.5 * 10 6, 2 * 10 6, and 6 * 10 6G/mol.
Preferably, the upper limit by the weight average molecular weight range of the quasi-interpenetrating networks of LPA and PDMA chain formation is about 20 * 10 6G/mol.The example of other upper limit comprises about 7 * 10 6, 10 * 10 6With 16 * 10 6G/mol.The specific examples of the weight-average molecular weight of a LPA/PDMA quasi-interpenetrating networks is about 6.4 * 10 6G/mol.
Preferably, quasi-interpenetrating networks has the composing type polymkeric substance, and wherein, the lower limit of the polydispersity index scope of every kind of composing type polymkeric substance is about 1.The example of other lower limit comprises about 1.05 and 1.1.
Preferably, quasi-interpenetrating networks has the composing type polymkeric substance, and wherein, the upper limit of the polydispersity index scope of every kind of composing type polymkeric substance is about 1.8.The example of other upper limit comprises about 1.3 and 1.5.
In preferred specific embodiment, the lower limit of the rotation radius scope of LPA in the quasi-interpenetrating networks and PDMA is less than about 10nm greatly.The example of other lower limit comprises about 15nm, 40nm, 80nm and 150nm.
Preferably, the upper limit of the rotation radius scope of LPA in the quasi-interpenetrating networks and PDMA approximately is 320nm.The example of other upper limit comprises about 165nm, 210nm and 280nm.One example of the rotation radius in the quasi-interpenetrating networks is about 153nm.
The overlapping concentration of quasi-interpenetrating networks in 1 * TTE that is made of LPA and PDMA chain approximately is 5 * 10 -4To 3.0 * 10 -2G/m1.The concrete example of an overlapping concentration is about 5.5 * 10 -3G/ml.
The scope of the weight molecular weight of LPA is about 6 * 10 6To 7 * 10 6In the time of between the g/mol, the scope of LPA content and PDMA content ratio is between about 10: 1 to 15: 1.
By LPA at first is provided main skeleton, allow LPA and PDMA under high polymer concentration, to mix, can prepare homogeneous, gelatinous solution thus, thereby form quasi-interpenetrating networks.Even LPA and the PDMA unmixing that becomes under the higher concentration condition, and macroscopic being separated appears in the suitable time, this method also can be implemented.
When LPA concentration is higher, as: greater than about 4.0 * 10 -2G/ml, PDMA and the LPA unmixing basically that becomes.But in the specific embodiment in the present invention, quasi-interpenetrating networks is formed by random copolymers and the homopolymer PDMA of AM and DMA.(AM/DMA's) random copolymers under high total polymer concentration and the condition that do not occur being separated (with PDMA) form quasi-interpenetrating networks possibility be provided.
In described specific embodiment, by using any suitable polymerization process, in damping fluid with acrylamide and DMAA monomer polymerization.Radical polymerization is preferred polymerization process.In case reach overlapping concentration in the buffered soln, then by the main skeleton of AM and DMA random copolymers chain formation random copolymers.The overlapping concentration of random copolymers approximately is 3 * 10 in the buffered soln -3To 4.0 * 10 -2G/ml.Another lower limit of scope described in the buffered soln is 5.0 * 10 -3G/ml.Another upper limit of scope described in the damping fluid is 3.0 * 10 -2G/ml.(specific examples as the overlapping concentration in 1 * TTE) is about 1.0 * 10 to buffered soln -2G/ml.
Preferably, the lower limit of weight average molecular weight range that constitutes the AM/DMA random copolymers chain of main skeleton is about 0.05 * 10 6G/mol.The example of other lower limit comprises about 0.1 * 10 6With 0.3 * 10 6G/mol.
Preferably, the upper limit of weight average molecular weight range that constitutes the AM/DMA random copolymers chain of main skeleton is about 2 * 10 6G/mol.The example of other upper limit comprises 0.5 * 10 6With 1 * 10 6G/mol.
Preferably, the lower limit of the ratio ranges of AM content and DMA content approximately is 5: 1 in quasi-interpenetrating networks.Other lower limit example comprises 10: 1 and 20: 1.Preferably, the upper limit of the ratio ranges of AM content and DMA content approximately is 50: 1 in quasi-interpenetrating networks.The example of other upper limit comprises 30: 1 and 40: 1.
Preferably, in the buffered soln rotation radius of AM/DMA random copolymers approximately between 10nm between the 80nm.The example of the lower limit of this scope is about 10nm, 15nm and 30nm.The example of the upper limit of this scope is about 40nm, 55nm and 80nm.Preferably, the polydispersity index of this random copolymers is between about 1.1 to 2.
By using any suitable polymerization process, when the main skeleton of random copolymers exists, can prepare the PDMA chain by polymerization DMA.Radical polymerization is preferred polymerization process.
Preferably, the lower limit of the weight average molecular weight range of the composing type polymkeric substance of quasi-interpenetrating networks and multipolymer approximately is 0.1 * 10 6G/mol, wherein this quasi-interpenetrating networks is by AM/DMA multipolymer and PDMA polymer formation.Other example of lower limit comprises about 0.5 * 10 6G/mol and 0.8 * 10 6G/mol.
Preferably, the upper limit of the weight average molecular weight range of the composing type polymkeric substance of quasi-interpenetrating networks and multipolymer is greater than 2 * 10 6G/mol, wherein this quasi-interpenetrating networks is made of AM/DMA multipolymer and PDMA polymkeric substance.Other example of the upper limit comprises about 1 * 10 6G/mol to 2 * 10 6G/mol.
Preferably, the lower limit of the rotation radius scope of this polymkeric substance and multipolymer approximately is 15nm.Other example of lower limit comprises about 40nm and 50nm.
Preferably, the upper limit of the range of turn radius of this polymkeric substance and multipolymer approximately is 80nm.Other example of the upper limit comprises about 55nm and 65nm.
Preferably, the polydispersity index of random copolymers and PDMA polymkeric substance is between about 1.1 to 2.One specific examples of polydispersity index is about 1.6.What it should be noted that is: there is two types polymkeric substance, and every kind of polydispersity index that oneself is all arranged wherein, this has widened the polydispersity index of quasi-interpenetrating networks, and is especially big when different at the molecular weight of two kinds of polymkeric substance.
In the optional specific embodiment in the present invention, the method for preparing quasi-interpenetrating networks can realize by mixed solution method.In the method, the polymkeric substance of formation quasi-interpenetrating networks and/or multipolymer requirement can be compatible with each other in the homogeneous of mixture of polymers, single-phase zone.
The independent solution of LPA and PDMA is provided in the method.One of solution in these solution, preferred PDMA solution is rarer than LPA solution.The example that is used for the solvent of these solution comprises water and buffering solution.The example of suitable buffered soln comprises Tris-TAPS-EDTA (TTE), Tris-boric acid-EDTA (TBE) and Tris-Glacial acetic acid-EDTA (TAE).Two kinds of polymkeric substance are thoroughly mixed, for the polymer chain IPN provides possibility.Two kinds of polymers solns are mixed with two kinds of solution that are higher than overlapping concentration does not far away have effectiveness.
The concentration of LPA solution approximately is 1 to 15 times of PDMA strength of solution.In the LPA specific embodiment identical with PDMA concentration, the volume of PDMA correspondingly reduces.The volume of LPA solution approximately is 1 to 50 times of PDMA liquor capacity.In the LPA specific embodiment identical with the PDMA volume, the concentration of PDMA correspondingly reduces.
In the substep mode PDMA solution is added in the LPA solution.After each the adding, with the solution thorough mixing that obtains.Based on the purpose in this specification sheets, the substep mode means that the part of PDMA solution total amount is added in the LPA solution, and after every part adds, with the solution thorough mixing that obtains.The example that is added into part comprises 1/3rd PDMA solution, 1/4th PDMA solution, 1/5th PDMA solution, 1/10th PDMA solution and twentieth PDMA solution.
In this specific embodiment, preferably, the lower limit of the weight average molecular weight range of LPA approximately is 0.05 * 10 6G/mol.The example of other lower limit comprises about 0.1 * 10 6, 0.3 * 10 6, and 1 * 10 6G/mol.
Preferably, the upper limit of the weight average molecular weight range of LPA approximately is 25 * 10 6G/mol (or high as far as possible).The example of other upper limit comprises about 4 * 10 6, 6 * 10 6, 7 * 10 6, 8 * 10 6, 10 * 10 6, 15 * 10 6With 20 * 10 6G/mol.One example of LPA weight-average molecular weight is 6.4 * 10 6G/mol.
LPA is dissolved in the aqueous solution, as water, or buffered soln.The example of suitable reducing comprises TTE, TBE and TAE.One example of preferred buffered soln is 1 * TTE+7M urea buffer solution.The LPA/ buffer agent solution, as LPA/TTE solution, the preferably about 1.0%g/ml of the lower limit of its concentration range.Other example of lower limit comprises 4.0% and 6.0%g/ml.The LPA/ buffer agent solution, as LPA/TTE solution, the preferably about 12.0%g/ml of the upper limit of its concentration range.Other example of the upper limit comprises 8.0% and 10.0%g/ml.Along with the increase of concentration, the mesh of quasi-interpenetrating networks size reduces.One example of LPA/TTE strength of solution is about 2.6%g/ml.
Preferably, the lower limit of the weight average molecular weight range of PDMA approximately is 100,000g/mol.The example of other lower limit comprises about 300,000 and 500,000g/mol.Preferably, the upper limit of the weight average molecular weight range of PDMA approximately is 25 * 10 6G/mol.The example of other upper limit comprises about 1 * 10 6, 3 * 10 6With 10 * 10 6G/mol.An example of PDMA weight-average molecular weight is 470,000g/mol.
PDMA is dissolved in the aqueous solution, as water or buffered soln.The example of suitable buffer reagent comprises TTE, TBE and TAE.The example of preferred buffered soln is 1 * TTE+7M urea buffer solution.The PDMA/ buffer agent solution, as PDMA/TTE solution, its concentration range preferably between about 0.1%g/ml between the 3.0%g/ml.An example of the concentration of PDMA/TTE solution is about 1.6%g/ml.
In the quasi-interpenetrating networks for preparing by this method, LPA approximately is 3.0 to 15.0 times of PDMA.Such as, quasi-interpenetrating networks can comprise about 85wt%LPA and 15wt%PDMA.
Solution mixing method also can be used for preparing the quasi-interpenetrating networks of AM/DMA random copolymers and PDMA.The independently buffered soln of preparation AM/DMA random copolymers and PDMA.The example of suitable buffered soln comprises TTE, TBE and TAE., a kind of solution in these solution, preferred PDMA solution is rarer than random copolymers solution.This polymkeric substance and copolymerization chain thorough mixing are for this chain IPN provides possibility.
The concentration of the random copolymers solution of AM/DMA approximately is 1 to 50 times of PDMA strength of solution.In this multipolymer specific embodiment identical with PDMA concentration, the volume of PDMA correspondingly reduces.The volume of this random copolymers solution approximately is 1 to 20 times of PDMA liquor capacity.In this multipolymer specific embodiment identical with the PDMA volume, the concentration of PDMA correspondingly reduces.The concentration of this multipolymer/buffer agent solution approximately is 5.0 to 20.0%g/ml, preferably approximately 7% to 12%g/ml.The concentration of PDMA/ buffer agent solution approximately is 0.1 to 1.0%g/ml.
, PDMA solution is joined in the random copolymers solution about the description of LPA and PDMA as preamble in the substep mode.
In this specific embodiment, preferably, the lower limit of the weight average molecular weight range of this random copolymers approximately is 0.05 * 10 6G/mol.The example of other lower limit comprises about 0.1 * 10 6And 0.3 * 10 6G/mol.Preferably, the upper limit of the weight average molecular weight range of this random copolymers approximately is 2 * 10 6G/mol (or high as far as possible, but in acceptable range of viscosities).The example of other upper limit comprises about 0.5 * 10 6, 1 * 10 6With 1.5 * 10 6G/mol.
Preferably, the lower limit of the weight average molecular weight range of PDMA approximately is 50,000g/mol.The example of other lower limit comprises 100,000 and 200,000g/mol.The upper limit of the weight average molecular weight range of PDMA approximately is 25 * 10 6G/mol.The example of other upper limit comprises 500,000,1 * 10 6, 3 * 10 6, 5 * 10 6With 10 * 10 6G/mol.
By the quasi-interpenetrating networks that this method prepares, its random copolymers than PDMA approximately up to 50 times.
The quasi-interpenetrating networks for preparing in all specific embodiments among the present invention can be used as the separating medium of charged molecule class material, as the DNA/ analysis of protein on capillary electrophoresis or the chip.The example of biomolecules comprises nucleic acid molecule and protein.
Under the situation of not considering instrumental variables, with regard to given polymkeric substance, the optimization of separation condition that is used for the capillary electrophoresis of dna sequence analysis depends on three principal elements: polymer concentration, sample injection rate and strength of electric field.The concentration affects of polymkeric substance is to resolving power, working hour with read long in the separating medium.Concentration is high more, and resolving power is high more, but concentration is low more, and viscosity is low more, separates sooner, reads longer.Optimum concn should be under alap concentration conditions, and resolving power is enough high.The sample injection rate has influence on resolving power and signal/noise ratio.For realizing best effect, it is minimum that injection rate should keep, so that high resolving power and sufficiently high signal/noise ratio to be provided.Strength of electric field should be suitably high, such as about 150V/cm.With regard to regard to small segment, strength of electric field is high more, separates fasterly, and resolving power is higher.But if strength of electric field is too high, bigger segmental resolving power will reduce, and reads long the shortening simultaneously.
As the DNA separating medium, quasi-interpenetrating networks successfully combines the dynamic coating ability of PDMA and the wetting ability of LPA.Simultaneously, because the part uncompatibility of these two kinds of polymeric systems, LPA and PDMA chain all obtain surpassing the stretching, extension of its natural conformation.This stretching, extension has improved the efficient of quasi-interpenetrating networks as separating medium, will be used to obtain the employed polymer materials amount of desirable mesh size simultaneously and be reduced to minimum.Do not increase other polymer-coated step by using exposed kapillary and just can obtain isolation resolution efficiently.
The quasi-interpenetrating networks that comprises LPA and PDMA is as separating medium, and its performance is subjected to the influence of several variablees.Such as, as mentioned before, the molecular weight of LPA and molecular weight distribution thereof play an important role in the dna sequencing analysis.The LPA molecular weight is high more, and range of molecular weight distributions is narrow more simultaneously, and its resolving power is high more, and separating power is stable more simultaneously.
In addition, with respect to the molecular weight of LPA, the ratio of LPA and PDMA has influence on separating effect.With regard to each weight molecular weight scope of LPA, but LPA:PDMA ratio optimized Separation effect.Such as, approximately be 6 * 10 with regard to weight-average molecular weight 6To 7 * 10 6LPA, the scope of LPA:PDMA ratio was between about 10: 1 to 15: 1 o'clock the bests.Even total polymer concentration is quite low, if PDMA content further increases, may cause the microphase-separated of this polymeric system, be unfavorable for separating effect.
Shown in Figure 20 and 21, under the working conditions of the best, by using 2.0w/v%LPA and PDMA, the LPA:PDMA ratio be 10: 1 as separating medium, at 60 ℃, in about 35 minutes, can reach more than 0.50 for the single base resolving power of 858 bases, be more than～0.3 for single base resolving power of 1000 bases.It should be noted that: LPA and PDMA total molecular weight are high more, and performance can be further enhanced.
Connect because this polymkeric substance does not carry out chemistry by linking agent, so this quasi-interpenetrating networks can easily be removed from kapillary.Therefore, if necessary, after each electrophoresis work, these polymers solns can be replaced.Such feature makes the separating medium among the present invention be highly suitable for automatic capillary electrophoresis.
The key point of preparation quasi-interpenetrating networks is: 1) select the most effective isolating polymer; 2) this isolating polymer and other mixed with polymers is even, this other polymkeric substance can dynamically be coated with inwall capillaceous or separating chips; And 3) every kind of polymkeric substance all is higher than the overlapping concentration of self.Therefore, main skeleton polymer should have system allowed the high polymers ratio in the total polymer concentration concentration of the decision of required mesh size (promptly by).This coated polymeric should lack as far as possible, but need be higher than the overlapping concentration of self.With regard to the separating medium that is used for sequencing analysis,, be easy to satisfy this requirement because total polymer concentration is relatively low.With regard to the DNA analysis of carrying out on the chip, promptly for using short passage length (channel length) and less charged molecule size to carry out real-time analysis, the mesh size is littler, and required total polymer concentration becomes higher.Under this concentration conditions, even if for miscible LPA when the low copolymer concentration and PDMA, also can occur being separated.Based on this, when total polymer concentration was higher, the consistency of the polymkeric substance in the quasi-interpenetrating networks can be regulated by utilizing the AM/DMA random copolymers, wherein, regulate the content of DMA in this multipolymer, make PDMA in the desired concn scope, become miscible with the random copolymers of AM/DMA.The quasi-interpenetrating networks separating medium should use the method among the present invention thoroughly to mix, and promptly carries out in-situ polymerization by a kind of polymkeric substance in other main skeleton polymer, or passes through the method for fractional steps.For obtaining repeatable result, mixing solutions should keep homogeneous on molecular level, but this does not depend on visual observation.
Embodiment
Embodiment 1
Material and method
Ammonium persulphate (APS), three (methylol) aminomethane (Tris), N-three-(methylol) methyl-3-amino propane sulfonic acid (TAPS), ethylenediamine tetraacetic acid (EDTA) (EDTA) and acrylamide available from Sigma company (St.Louis, MO, USA).Acrylamide is by twice recrystallization of chloroform.(AOT Fluka) is dissolved in the methyl alcohol to remove salinity, filtration then with sulfo-succinic acid two (2-ethylhexyl) ester.Then with filtrate and sherwood oil one oscillates, so that the extracting organic impurity.Remove subsequently and desolvate, in the presence of Vanadium Pentoxide in FLAKES, in 50 ℃ of vacuum-drying throw outs.Diisopropyl azodicarboxylate (AIBN) is by twice recrystallization of ethanol.N, N, N ＇, N ＇-tetramethylene-diamine (TEMED) available from Fisher Scientific (Pittsburgh, PA).(Warrington, PA USA), distill under 80 ℃/20mmHg before using DMA available from Polysciences.Anode and negative electrode working buffer liquid all are 1 * TTE (50mMTris/50mM TAPS/2.0mM EDTA).The same with isolation medium, negative electrode working buffer liquid also comprises-7M urea.Respond and solution in the water that uses all through the Milli-Q water purification system (Millipore, Bedford, MA, USA) deionization is to 18.2M Ω.
Prepare LPA by conversed phase micro emulsion copolymerization
Table 1 has been listed typical LPA preparation method.Polyreaction is in nitrogen, and 45 ℃, stirring velocity is to carry out under the condition of 350rpm.Except that AIBN solution, all the components all is placed in the 150ml four neck round-bottomed flasks.This round-bottomed flask is equipped with mechanical stirrer, reflux exchanger, thermometer, nitrogen inlet pipe, and feed hopper.Before AIBN solution added, reactive system was used nitrogen purging one hour.Polymerization precipitates in excessive propanone after finishing, continuous washing, filtration and 50 ℃ of vacuum-dryings, thus reclaim polyacrylamide.
Table 1 polyacrylamide polymerization aMethod
Figure S04827066420060404D000261
A): polymerization time: 2h
B): polymerization temperature: 60 ℃
The preparation quasi-interpenetrating networks
LPA is dissolved in the water in the 100ml four neck round-bottomed flasks, and this round-bottomed flask is equipped with mechanical stirrer, reflux exchanger, thermometer, nitrogen inlet pipe, and feed hopper.After adding DMA, reactive system is used nitrogen purging one hour, adds APS and TEMED then.Polymerization precipitates in excessive propanone after finishing, continuous washing, filtration and 50 ℃ of vacuum-dryings subsequently, thus reclaim quasi-interpenetrating networks.Table 2 has been listed the method for typical preparation based on the quasi-interpenetrating networks of LPA and PDMA.
Table 2 is based on the preparation method of the quasi-interpenetrating networks of acrylamide and DMAA
Polymerization temperature: 0 ℃
Polymerization time: 24h
The feature of polymkeric substance
Utilize Ubbelohde viscometer determining 1M NaNO 3The intrinsic viscosity of LPA [η] in the aqueous solution, the intrinsic viscosity of this LPA during by 30 ℃ of extrapotation calculating, zero-dose.Formula below using calculates viscosity-molecular-weight average (Mv) (table 1) of synthetic LPA: [η]=3.73 * 10 -2Mv 0.66Cm 3/ g.
Pass through AC250- 1H-NMR measures the composition of quasi-interpenetrating networks, and it is with D 2O is as solvent.The ratio (table 2) of LPA and PDMA is to be positioned at 2.1 and the integration peak area ratio estimation at the peak of 2.6ppm in the quasi-interpenetrating networks.
The order-checking chemistry
Use has
Figure S04827066420060404D000271
The ABI PRISM of archaeal dna polymerase TMDye Primer (21M 13forward) Cycle Sequencing Ready Reaction Kit, (CA USA) carries out sequencing reaction to the F S on pGEM3Zf (+) double-stranded template for PE Biosystems/Perkin-Elmer, Foster City.Use the primer of FAM mark and the sequencing reaction that C end mixture carries out single dye marker.Dna sequencing utilizes GeneAmp PCR System2400 (PE Biosystems/Perkin-Elmer) to carry out, and cycling condition is as follows: 95 ℃ 10 seconds, 50 ℃ 5 seconds and 70 ℃ of 15 circulations of 1 minute, 95 ℃ of 10 seconds and 70 ℃ of 15 circulations of 1 minute subsequently.Reaction product through the ethanol sedimentation purifying, subsequently with 95% and twice of 75%v/v washing with alcohol, 20 μ l deionized formamides in suspend again.
The fluoroscopic examination of induced with laser
Argon ion laser is used to form the incident power of excitation beam and the about 5mW of λ=488nm.This laser beam through focal length be the lens focus of 25cm, by dichroic mirror (550DRLP; Omega Optical, Brattleboro, VT) reflection uses 10 * object lens to focus on once more on the interior point of split tunnel then.(NJ) preceding, (Brattleboro VT) filters for 530DF30, Omega Optical by bandpass filter for Hamamatsu R928, Hamamatsu Corporation entering photomultiplier through the fluorescence of dichroic mirror.Be to obtain the kapillary image, this device also be connected with charge coupled device (CCD) pick up camera (SONY SSC-M350, SONY Corporation, New York, NY).The white light beam that light-emitting device on the microscope sends illuminates kapillary.Then, it is by object lens focusing, by slipping into/skid off mirror (slide in-and-out mirror) reflection, and (D.0Industries, Rochester NY), detects by ccd video camera subsequently by the amplification of Zoom 6000 systems.(KarlZeiss, Melville NY) provide the method that obtains superior optical quality and rapid alignment with ccd video camera to microscope.
The CE step
1. at the homemade CE instrument in laboratory
Use has the fused quartz kapillary (PolymicroTechnologies.Phoenix AZ) of the ID/OD of 75/365 μ m.By opening detection window at distance positive terminal 8cm place with blade strip off polyimide coating.Kapillary simply washs 10min with 1M HC1.Use gastight syringe that separating medium is injected kapillary.Before each electrophoresis work, capillary column is debugged under 300V/cm strength of electric field, until current stabilization (generally about 10min).In electronic mode the DNA sample is injected kapillary.Concrete injection condition, concrete length and deposition condition have been listed in the description of drawings.
2. at ABI-310Step is (1) together, but follows the routine operation of ABI process.
Embodiment 2: variable
The influence of working conditions
Working conditions is injected width, effective column length and applied electric field intensity as DNA, and is very big to the performance impact of quasi-interpenetrating networks in DNA separates.For relatively, calculate the segmental resolving power of selected DNA (R) according to following formula: R=2 (t 2-t 1)/[1.699 (w 2+ w 1)], wherein t and w represent the full peak width of transition time and 1/2 maximum height respectively.
In CE, the DNA sample generally is to inject capillary column by electronic injection system.The amount of initial DNA bandwidth or the DNA that is injected into is regulated and control at interval by regulating injecting voltage and injection length.
Figure 14 shows the variation of using the resolving power of quasi-interpenetrating networks under different DNA injecting voltages among the embodiment 1.Keep injection length interval (10s) constant, utilize formula (2) calculating injecting voltage to be: the isolating resolving power of base fragment when 25V/cm, 75V/cm and 100V/cm, with respect to fragment length resolving power is mapped (Figure 14) simultaneously, wherein detected base fragment length is: 103/104,158/159,186/187,216/217,280/281,310/311,357/358,411/413,716/720 and 833/836.(wherein the resolving power of 411/413 base pair divided by the resolving power of 2,716/720 base pairs divided by the resolving power of 4 and 833/836 base pair divided by 3).Compare with 25V/cm with injecting voltage 100V/cm, resolving power was the highest when injecting voltage was 75V/cm.Higher injecting voltage (being higher than 75V/cm) not only increases initial DNA bandwidth, and causes hangover before the peak.The generation of this phenomenon be because: dna fragmentation is in sample buffer and the excessive accumulation of separating medium intersection.Injecting voltage is too high, and injection length is long, will cause that some dna fragmentations are imported into separating medium before applying operating voltage.Bandwidth increases, and type distortion in peak can reduce separating effect.Injecting voltage is lower, can shorten initial bandwidth, thereby improves resolving power.But if brownout, signal/noise ratio will reduce.Injection length is at interval to the same injecting voltage of the isolating influence mode of DNA.
Under 75V/cm, injection length 10s, separating effect best (not providing data).Injecting voltage and injection length are closely related with initial DNA bandwidth at interval.When other working conditions, when remaining unchanged, there is a best of breed as column length, temperature and applied electric field intensity.
Another is a column length to the parameter that DNA separation tool has a significant impact.The variation of resolving power when Figure 15 illustrates effective column length and is 40cm and 35cm.The column length of 40cm is higher than the column length resolving power of 35cm.In carry out the determined dna sequence analysis with CE, 13cm is used with different separating mediums together to the column length of 60cm.In general, there is a minimum column length; Column length is littler than minimum column length, then can not obtain gratifying separation; It is longer that column length can cause DNA to read than length usually, but the working hour is short; Can realize sharp separation by reducing column length, but reduce long and the resolving power read of DNA simultaneously.
The applied electric field intensity effect to the speed of dna sequencing, read long and resolving power.Figure 16 is when applied electric field intensity is 200V/cm and 150V/cm, pGEM3Zf (+) electrophorogram (C-mix) after 581 bases.Compare with higher applied electric field intensity (200V/cm), lower applied electric field intensity (150V/cm) make DNA read longer, resolving power is higher and transition time is longer.
Temperature effect DNA separates.Usually, column temperature suitably increases (being lower than 50 ℃), can improve separating effect and velocity of separation, and this also helps the big segmental separation of DNA.When the quasi-interpenetrating networks that uses LPA and PDMA to constitute during, can observe very close result (data do not provide) as the DNA separating medium.
The influence of LPA and PDMA ratio
Figure 17 shows the separation of dna sequencing sample, and it is 1.62: 1 that this separation is to use LPA and PDMA ratio, and 3.33: 1, the quasi-interpenetrating networks of 6.33: 1 and 10.9: 1 was as separating medium.In uncoated kapillary, be 6.4 * 10 with molecular weight 6The LPA of g/mol is as the DNA separating medium, and the non-constant of separation efficiency is shown in Figure 17 a.Can significantly improve separating effect by adding very small amount of PDMA formation quasi-interpenetrating networks, shown in Figure 17 e, wherein, LPA and PDMA ratio are 10.89: 1.But the further increase of PDMA amount can weaken separating effect.Shown in Figure 17 b-d, when LPA and PDMA ratio when drop to 1.62: 1 at 6.33: 1, the corresponding reduction of isolation resolution.
In Figure 17, the molecular weight of LPA very high (6.4 * 10 6G/mol).Being formed on of quasi-interpenetrating networks that contains a small amount of PDMA (LPA:PDMA is 10.9: 1) optimized this network under the situation about not being separated.In addition, PDMA provides enough strong dynamic coating ability, can suppress electroosmotic flow.PDMA content increases (LPA:PDMA is 6.33: 1) and can cause separation efficiency poorer.And PDMA content further increase can cause microphase-separated, and this can destroy the performance of quasi-interpenetrating networks, shown in Figure 17 b and 17c.
The influence of LPA molecular weight
In order to develop the quasi-interpenetrating networks that is applicable to that dna sequencing is analyzed, use the high as far as possible LPA of molecular weight to form main skeleton, then with a small amount of PDMA monomer polymerization, with the formation of optimizing quasi-interpenetrating networks and dynamic coating ability is provided.Figure 18 shows the LPA molecular weight to the isolating influence of DNA.When keeping LPA:PDMA ratio almost constant and lower (less than 10: 1 consistencies with maintenance LPA and PDMA), the molecular weight of LPA is high more, and resolving power is good more and read long long more.The main skeleton of the quasi-interpenetrating networks that is formed by large-sized LPA provides nearly all screening capacity.The LPA chain has been expanded in the existence of IPN PDMA chain, and, make separating medium more effective.
The influence of LPA molecular weight distribution
As shown in figure 18, higher LPA molecular weight can produce better resolving power and longer DNA and reads long in the quasi-interpenetrating networks.In order to increase the molecular weight of LPA, can adopt different synthetic methods.In one approach, adopt the scheme of emulsifier-free to come polymerization LPA (sample AO, table 1).In another approach, adopt the scheme of conversed phase micro emulsion copolymerization to come polymerization LPA (sample A4, table 1).All successfully synthesize fragment greater than 9 * 10 with two kinds of methods 6High molecular LPA.Such LPA is used to form the quasi-interpenetrating networks of the PDMA with similar quantity, and its performance in DNA separates as shown in figure 19.After same PDMA (Figure 19 b) formed quasi-interpenetrating networks, the separation efficiency was better with conversed phase micro emulsion copolymerization method synthetic LPA.Other all conditions is as the method for LPA molecular weight, LPA:PDMA ratio and formation quasi-interpenetrating networks, much at one.
Dna sequencing under the optimal conditions
In order to improve the isolating treatment capacity of DNA, reading long long sharp separation is a very practical method.Working conditions below adopting: effective column length is 34m, and operating voltage is 225V/cm, carries out DNA at 75V/cm and injects, and the time is 8s, and temperature is 45 ℃.Quasi-interpenetrating networks has following parameter: the 2.5w/v% (M of LPA vBe 4.53 * 10 6G/mol), the LPA/PDMA ratio is 10.81: 1.Use the Fam mark-the 21M13 primer forms the segmental electrophoresis spectrogram of determined dna sequence that C holds single dye marker on pGEM-3Zf (+) template, as shown in figure 15.At room temperature, in the time of realizing up to 617 bases, single base resolving power is 0.63, and single base resolving power is 0.3 during up to～1000 bases.It should be noted that 50 ℃ of service temperatures high more (〉), it is short more to have a required working hour of suitable resolving power.
Embodiment 3
Polymerization PDMA is to generate quasi-interpenetrating networks in LPA
(a) specimen preparation:
The employing conversed phase micro emulsion copolymerization method acrylamide monomer of purifying aggregates into LPA.In the main skeleton of LPA, the DMAA monomer is carried out radical polymerization, form quasi-interpenetrating networks.
(b) the laser light scattering preparation of solution:
Prepare two kinds and be dissolved in 1 * TTE buffered soln, concentration is respectively 7.77 * 10 -2G/ml and 1.20 * 10 -2The LPA of g/ml and the stock solution of quasi-interpenetrating networks.This stock solution is kept in the volumetric flask of 50ml, vibrating at least 7 to 10 days on the shaking mixer at a slow speed, with the solution of preparation homogeneous.Be used for by dilution stock solution preparation that scattering of light detects, six kinds of LPA solution and five kinds of quasi-interpenetrating networks solution of prescribed concentration.Every kind of diluent is passed through centrifugal (7700g (8000rpm), 6h) purification.The dustless solution in upper strata is used for detecting the feature of this solution scattering of light.
(c) scattering of light detects:
In scattering angle is 15 0To 139 0, under 25 ℃ of the temperature, use laboratory self-control laser light scattering spectrometer to carry out static state and dynamic light scattering detection.Laser output power is～500mW at 532nm wavelength place.Utilizing Brookhaven Instruments B 19000 digital correlation instrument to be determined at scattering angle is 30 0, 35 0, 40 0, 50 0, 60 0, 70 0With 90 0The time intensity-intensity autocorrelative function.
Fig. 1 and Fig. 2 show the static light scattering result of LPA and quasi-interpenetrating networks respectively.Fig. 3 and Fig. 4 show the dynamic light scattering result of LPA and quasi-interpenetrating networks respectively.Two peaks appear in Fig. 3, and one of them is at R hThe minor peaks at～15nm place, and another is at R hThe main peak at～80nm place.For quasi-interpenetrating networks, this minute sized composition can be removed in clarifying process.But the existence of this minor peaks may be the indication of the polymerization process of LPA, wherein may relate to two independent processes that molecular chain increases.Form the molecular chain of reduced size in the beginning step, form the molecular chain of large-size in the subsequent step.
(d) capillary electrophoresis
The preparation of quasi-interpenetrating networks separating medium:
In a 5ml bottle, the exsiccant quasi-interpenetrating networks is dissolved in 1 * TTE+7M urea buffered soln.Total liquor capacity generally is less than 2ml, and polymer concentration is about 2-2.5%g/ml.By standing over night, make dry sample be swelled into gelatinous solution after, on vortex mixer, this gelatinous solution is thoroughly mixed 20s, every day twice, all dissolve up to polymkeric substance.At least to spend 3-4 days time to prepare its uniform solution.Before using, (polymers soln) separating medium will carry out centrifugal, to remove bubble.
(e) CE electrophoresis:
Three kinds are used for the different instruments that CE detects:
(i) the homemade laser induced fluorescence detector in laboratory with water-cooled Argon ion laser, λ=488nm, it is used for the dna sequencing analysis of the primer of single dyestuff [FAM] mark and C end hybrid dna (pGEM-3Zf (+) from-21M13 upstream primer).
In Fig. 5, the sequencing analysis data show: the separation with 1000 bases of single base resolving power (only with the naked eye) can be finished in 30min.
Be used for the comparison of the commerical prod of isolating quasi-interpenetrating networks of DNA and various polymeric medias
Fig. 6 shows DNA (pGEM-3Zf (+) from-21M13 upstream primer) sequencing analysis data.Recommend according to Amersham Biosciences company, under 44 ℃ and 150V/cm, carry out capillary electrophoresis in MegaBACE LPA gel/1 * MegaBACE damping fluid.Fig. 7 shows the sequencing analysis data of same DNA, but is under 50 ℃ and 200V/cm, carries out CE in medium POP-6 (Applied Biosystems)/1 * TTE damping fluid, and this is the CE routine work condition of POP6.
As a comparison, can be with the transition time of the DNA sample of 800b as isolating velocity standard, utilize the quasi-interpenetrating networks can be at 26.3min (t as the separation of separating medium 800, IPN) in finish (Fig. 5), and utilize MegaBACE LPA gel to be separated in 89min (t as separating medium 800, LPA) in just can finish (Fig. 6).
The velocity of separation of DNA is than fast about 3 times of the velocity of separation of DNA in MegaBACE LPA gel in the quasi-interpenetrating networks medium.DNA in the POP6 medium separates can not reach 800b (Fig. 7).If the transition time of 590b can be used as the isolating velocity standard of DNA, using quasi-interpenetrating networks, MegaBACE LPA and POP6 so is respectively t as the transition time of separating medium 590, IPN=～22min, t 590, LPA=～70.5min and t 590, POP6=～89min.In quasi-interpenetrating networks the required time of the separation of 590bpDNA only be in MegaBACE LPA required time 1/3rd, in POP6 1/4th of required time.
The ABI310 genetic analyzer is a kind of single capillary genetic analyzer of automatization, and it is designed and is optimised, uses with order-checking and the fragment analysis of supporting wide region.Show the sequencing analysis performance with BigDyeTerminator v3.0 standard DNA.
Fig. 8 shows the terminal portions (base number from 600 to～1000) of the electrophoretic separation of BigDye v3.0.On ABI310, use quasi-interpenetrating networks medium through characterizing, can in 36min, finish the separation of 1000 bases of four look BigDye v3.0DNA.
Fig. 9 shows and utilizes POP6 medium and Module seq POP6 (1mL) E BigDye v3.0 to be carried out the blueness-G part of electrophoretic separation.860b (t 860, POP6) transition time be～61.5min to be the 860b (t that utilizes the quasi-interpenetrating networks separating medium 860, IPN) 1.9 times (Fig. 8) of transition time～32min.ABI310 can not use MagBACE LPA gel, because this gel viscosity height, and the pump pressure of ABI310 is not enough to detect.
When quasi-interpenetrating networks was used as separating medium, naked eyes can read long base identification (1ong base-call) (1007b) (Fig. 8) from the electrophoretic separation data, and when POP6 was used as separating medium, naked eyes can only read 860b (Fig. 9).
Homemade fluorescence (four looks) detector (Perkin E1mer Model SPCM-AQR-12-FC) with induced with laser of silicon avalanche photodiode in laboratory can be in order to carry out sequential analysis to BigDye Terminator v3.0.This detector is by SUNY engineering college (Engineering College) preparation of Stony Brook.As a comparison, CE carries out in the different medium of the quasi-interpenetrating networks and the product innovation POP7 of Applied Biosystems company.
In Figure 10, the base recognition data of DNA (BigDye Terminator v3.0) shows: under 60 ℃ and 304V/cm, when CE carried out in quasi-interpenetrating networks/1 * ABI damping fluid, the separation energy of 600 bases with single base resolving power was at～30.4min (t 600, IPN) in finish.The kapillary useful length is 50cm, kapillary ID/OD=50/361 (μ m).But when POP7/1 * ABI damping fluid during as separating medium, under 60 ℃ and 196V/cm, the best separating resulting that the separation of 600 bases of same DNA is obtained is at～63.3min (t 600, POP7) in.The ratio of kapillary useful length and ID/OD is respectively identical, at 50cm and 50/361 (μ m).Figure 11 is the base recognition data.In the quasi-interpenetrating networks medium, the DNA that separates the 600b with single base resolving power separates required time ratio required time in POP7 and lacks half.(instrument and base identification software are provided by the SUNY engineering college of StonyBrook.)
Embodiment 4
Form the solution blending means of quasi-interpenetrating networks
Prepare the diluting soln of two kinds of different polymkeric substance respectively.These two kinds of polymers solns are admixed together gradually, have the uniform solution mixture of the composition of prescribed concentration and polymeric constituent with formation.
LPA and PDMA are that the part compatible polymers is right.Use separating medium for preparation CE detects, make the LPA and the PDMA solution mixture of homogeneous by using the solution blending means.Prepare LPA/ buffer agent solution and PDMA/ buffer agent solution respectively.With LPA (M w=7.62 * 10 6G/mol) be dissolved in 1 * TTE+7M urea buffer solution, to be mixed with the solution of 2.57%g/ml.With PDMA (M w=470Kg/mol) be dissolved in 1 * TTE+7M urea buffer solution, to be mixed with the solution of 1.61%g/mL.After dissolving fully in the buffer reagent of every kind of polymkeric substance at himself, 300 μ lPDMA solution are progressively added in the 0.9ml LPA solution with 100 μ l aliquot.Behind each interpolation PDMA solution, solution mixture is mixed 20s on vortex mixer.The content of LPA and PDMA is respectively 83% and 17% in the solution mixture.Noticeable: when the PDMA molecular weight was 470K, concentration 1.61%g/mL had been higher than its C *Value.
Figure 12 shows utilization according to the LPA/PDMA quasi-interpenetrating networks of the embodiment 3 preparation CE result as separating medium.As a comparison, Figure 13 shows utilization according to the quasi-interpenetrating networks of the embodiment 2 preparation CE result as separating medium.Under same working conditions, the DNA separating resulting of two kinds of separating mediums is very close.Electrophoresis is to carry out under the 195.5V/cm at applied electric field, and the DNA injection length is 20s when 27V/cm.
Embodiment 5
A kind of solution blending means that generates quasi-interpenetrating networks
Starting material comprise the LPA of the about 7M of molecular weight; PDMA with the about 0.5M of molecular weight.Adopting several different methods as follows to prepare final total concn is 2～2.5-wt% (mixing solutions of 0.02～0.025g/mL).
A) concentration ratio (wt%) of preparation LPA/PDMA is 1.6/1 solution.The concentration of LPA solution is 0.0257g/mL, and the concentration of PDMA solution is 0.016g/mL.Adopt following each mode to mix the mixing solutions that final total concn is 2～2.5wt%.
Figure S04827066420060404D000371
*Suppose that volume and volume unit are constant.
B) concentration ratio (wt%) of preparation LPA/PDMA is 2/1 solution, and wherein the concentration of LPA solution is 0.028g/ml, and the concentration of PDMA solution is 0.014g/ml.Adopt following each mode to mix the mixing solutions that final total concn is 2～2.5wt%.
Figure S04827066420060404D000381
*Suppose that volume and volume unit are constant.

Claims (120)

1. the quasi-interpenetrating networks of a polymer chain, described polymer chain comprises:
(a) the linear polyacrylamide chain that exists with main skeleton form, the weight-average molecular weight of described polyacrylamide chain is 0.05 * 10 6To 25 * 10 6G/mol, rotation radius is that 10nm is to 350nm; And
(b) polydimethylacrylamiin chain, described polydimethylacrylamiin chain are in the presence of the main skeleton of linear polyacrylamide, by the preparation of polymerization DMAA,
Wherein, described linear polyacrylamide chain and polydimethylacrylamiin chain tangle each other, IPN, and wherein, described quasi-interpenetrating networks does not have chemically crosslinked.
2. network according to claim 1, wherein, the main skeleton of linear polyacrylamide is to form by conversed phase micro emulsion copolymerization.
3. network according to claim 1, wherein, described polydimethylacrylamiin is that polymerization forms by radical polymerization in the main skeleton of linear polyacrylamide.
4. network according to claim 1, wherein, the main skeleton of described linear polyacrylamide comprises the linear polyacrylamide in the buffered soln, wherein, the overlapping concentration that described linear polyacrylamide has is 5 * 10 -4To 4.0 * 10 -2G/ml.
5. network according to claim 1, wherein, the overlapping concentration of the main skeleton of described linear polyacrylamide in buffered soln is 1.2 * 10 -3G/ml.
6. network according to claim 1, wherein, the following of the weight-average molecular weight of described linear polyacrylamide is limited to 0.05 * 10 6G/mol.
7. network according to claim 1, wherein, the following of the weight-average molecular weight of described linear polyacrylamide is limited to 0.1 * 10 6G/mol.
8. network according to claim 1, wherein, the following of the weight-average molecular weight of described linear polyacrylamide is limited to 0.3 * 10 6G/mol.
9. network according to claim 1, wherein, the following of the weight-average molecular weight of described linear polyacrylamide is limited to 1 * 10 6G/mol.
10. network according to claim 1, wherein, the following of the weight-average molecular weight of described linear polyacrylamide is limited to 4 * 10 6G/mol.
11. network according to claim 1, wherein, the following of the weight-average molecular weight of described linear polyacrylamide is limited to 6 * 10 6G/mol.
12. network according to claim 1, wherein, the following of the weight-average molecular weight of described linear polyacrylamide is limited to 7 * 10 6G/mol.
13. network according to claim 1 wherein, is limited to 8 * 10 on the weight average molecular weight range of described linear polyacrylamide 6G/mol.
14. network according to claim 1 wherein, is limited to 10 * 10 on the weight average molecular weight range of described linear polyacrylamide 6G/mol.
15. network according to claim 1 wherein, is limited to 15 * 10 on the weight average molecular weight range of described linear polyacrylamide 6G/mol.
16. network according to claim 1 wherein, is limited to 25 * 10 on the weight average molecular weight range of described linear polyacrylamide 6G/mol.
17. network according to claim 1, wherein, the following 10nm that is limited to of the rotation radius scope of described linear polyacrylamide.
18. network according to claim 1, wherein, the following 15nm that is limited to of the rotation radius scope of described linear polyacrylamide.
19. network according to claim 1, wherein, the following 28nm that is limited to of the rotation radius scope of described linear polyacrylamide.
20. network according to claim 1, wherein, the following 55nm that is limited to of the rotation radius scope of described linear polyacrylamide.
21. network according to claim 1, wherein, the following 125nm that is limited to of the rotation radius scope of described linear polyacrylamide.
22. network according to claim 1, wherein, the following 150nm that is limited to of the rotation radius scope of described linear polyacrylamide.
23. network according to claim 1, wherein, the following 165nm that is limited to of the rotation radius scope of described linear polyacrylamide.
24. network according to claim 1 wherein, is limited to 180nm on the rotation radius scope of described linear polyacrylamide.
25. network according to claim 1 wherein, is limited to 210nm on the rotation radius scope of described linear polyacrylamide.
26. network according to claim 1 wherein, is limited to 250nm on the rotation radius scope of described linear polyacrylamide.
27. network according to claim 1 wherein, is limited to 350nm on the rotation radius scope of described linear polyacrylamide.
28. network according to claim 1, wherein, the polydispersity index of described linear polyacrylamide is 1.01 to 1.8.
29. network according to claim 28, wherein, the polydispersity index scope of described linear polyacrylamide following be limited to 1.01.
30. network according to claim 28, wherein, the following of the polydispersity index scope of described linear polyacrylamide is limited to 1.02.
31. network according to claim 28, wherein, the following of the polydispersity index scope of described linear polyacrylamide is limited to 1.05.
32. network according to claim 28, wherein, the following of the polydispersity index scope of described linear polyacrylamide is limited to 1.1.
33. network according to claim 28 wherein, is limited to 1.3 on the polydispersity index scope of described linear polyacrylamide.
34. network according to claim 28 wherein, is limited to 1.5 on the polydispersity index scope of described linear polyacrylamide.
35. network according to claim 28 wherein, is limited to 1.6 on the polydispersity index scope of described linear polyacrylamide.
36. network according to claim 28 wherein, is limited to 1.8 on the polydispersity index scope of described linear polyacrylamide.
37. the quasi-interpenetrating networks of the polymer chain of an entanglement, wherein said polymer chain comprises:
(a) linear polyacrylamide chain; With
(b) become entangled in the described linear polyacrylamide chain and the polydimethylacrylamiin chain of IPN in the linear polyacrylamide chain,
Wherein, the weight-average molecular weight of described linear polyacrylamide and polydimethylacrylamiin is 0.1 * 10 6G/mol to 20 * 10 6G/mol, rotation radius be 15nm to 320nm, wherein said quasi-interpenetrating networks does not have chemically crosslinked.
38. according to the described network of claim 37, wherein, the following of described weight average molecular weight range is limited to 0.1 * 10 6G/mol.
39. according to the described network of claim 37, wherein, the following of described weight average molecular weight range is limited to 0.5 * 10 6G/mol.
40. according to the described network of claim 37, wherein, the following of described weight average molecular weight range is limited to 2 * 10 6G/mol.
41. according to the described network of claim 37, wherein, the following of described weight average molecular weight range is limited to 6 * 10 6G/mol.
42., wherein, be limited to 7 * 10 on the described weight average molecular weight range according to the described network of claim 37 6G/mol.
43., wherein, be limited to 10 * 10 on the described weight average molecular weight range according to the described network of claim 37 6G/mol.
44., wherein, be limited to 16 * 10 on the described weight average molecular weight range according to the described network of claim 37 6G/mol.
45., wherein, be limited to 20 * 10 on the described weight average molecular weight range according to the described network of claim 37 6G/mol.
46. according to the described network of claim 37, wherein, the following 15nm that is limited to of described rotation radius scope.
47. according to the described network of claim 37, wherein, the following 40nm that is limited to of described rotation radius scope.
48. according to the described network of claim 37, wherein, the following 80nm that is limited to of described rotation radius scope.
49. according to the described network of claim 37, wherein, the following 150nm that is limited to of described rotation radius scope.
50., wherein, be limited to 165nm on the described rotation radius scope according to the described network of claim 37.
51., wherein, be limited to 210nm on the described rotation radius scope according to the described network of claim 37.
52., wherein, be limited to 280nm on the described rotation radius scope according to the described network of claim 37.
53., wherein, be limited to 320nm on the described rotation radius scope according to the described network of claim 37.
54. according to the described network of claim 37, wherein, the polydispersity index of described linear polyacrylamide and polydimethylacrylamiin is 1.0 to 1.8.
55. according to the described network of claim 37, wherein, the polydispersity index of described linear polyacrylamide and polydimethylacrylamiin is 1.6.
56. according to the described network of claim 37, wherein, the overlapping concentration of described network in buffered soln is 5.0 * 10 -4To 3.0 * 10 -2G/ml.
57. the quasi-interpenetrating networks of the polymer chain of an entanglement is prepared by following method, described method comprises:
(a) provide a kind of solution that comprises linear polyacrylamide and buffer reagent, wherein, the weight-average molecular weight of described linear polyacrylamide is 0.05 * 10 6To 25 * 10 6G/mol;
(b) provide a kind of solution that comprises polydimethylacrylamiin and buffer reagent, wherein, the weight-average molecular weight of described polydimethylacrylamiin is 100000 to 25 * 10 6G/mol;
(c) in the substep mode linear polyacrylamide/buffer agent solution and polydimethylacrylamiin/buffer agent solution are mixed, wherein, the concentration of described linear polyacrylamide/buffer agent solution is 1 to 15 times of polydimethylacrylamiin/buffer agent solution concentration, and the volume of described linear polyacrylamide/buffer agent solution is 1 to 50 times of polydimethylacrylamiin liquor capacity; Wherein, prepare by entanglement, the linear polyacrylamide of IPN and the quasi-interpenetrating networks that the polydimethylacrylamiin polymer chain constitutes, wherein, described interpenetrating(polymer)networks do not have chemically crosslinked.
58. according to the described quasi-interpenetrating networks of claim 57, wherein, the following of the weight average molecular weight range of described linear polyacrylamide is limited to 0.05 * 10 6G/mol.
59. according to the described quasi-interpenetrating networks of claim 57, wherein, the following of the weight average molecular weight range of described linear polyacrylamide is limited to 0.1 * 10 6G/mol.
60. according to the described quasi-interpenetrating networks of claim 57, wherein, the following of the weight average molecular weight range of described linear polyacrylamide is limited to 0.3 * 10 6G/mol.
61. according to the described quasi-interpenetrating networks of claim 57, wherein, the following of the weight average molecular weight range of described linear polyacrylamide is limited to 1 * 10 6G/mol.
62., wherein, be limited to 4 * 10 on the weight average molecular weight range of described linear polyacrylamide according to the described quasi-interpenetrating networks of claim 57 6G/mol.
63., wherein, be limited to 6 * 10 on the weight average molecular weight range of described linear polyacrylamide according to the described quasi-interpenetrating networks of claim 57 6G/mol.
64., wherein, be limited to 7 * 10 on the weight average molecular weight range of described linear polyacrylamide according to the described quasi-interpenetrating networks of claim 57 6G/mol.
65., wherein, be limited to 8 * 10 on the weight average molecular weight range of described linear polyacrylamide according to the described quasi-interpenetrating networks of claim 57 6G/mol.
66., wherein, be limited to 10 * 10 on the weight average molecular weight range of described linear polyacrylamide according to the described quasi-interpenetrating networks of claim 57 6G/mol.
67., wherein, be limited to 15 * 10 on the weight average molecular weight range of described linear polyacrylamide according to the described quasi-interpenetrating networks of claim 57 6G/mol.
68., wherein, be limited to 20 * 10 on the weight average molecular weight range of described linear polyacrylamide according to the described quasi-interpenetrating networks of claim 57 6G/mol.
69. according to the described quasi-interpenetrating networks of claim 57, wherein, the following 100000g/mol that is limited to of the weight average molecular weight range of described polydimethylacrylamiin.
70. according to the described quasi-interpenetrating networks of claim 57, wherein, the following 300000g/mol that is limited to of the weight average molecular weight range of described polydimethylacrylamiin.
71. according to the described quasi-interpenetrating networks of claim 57, wherein, the following 500000g/mol that is limited to of the weight average molecular weight range of described polydimethylacrylamiin.
72., wherein, be limited to 1 * 10 on the weight average molecular weight range of described polydimethylacrylamiin according to the described quasi-interpenetrating networks of claim 57 6G/mol.
73., wherein, be limited to 3 * 10 on the weight average molecular weight range of described polydimethylacrylamiin according to the described quasi-interpenetrating networks of claim 57 6G/mol.
74., wherein, be limited to 10 * 10 on the weight average molecular weight range of described polydimethylacrylamiin according to the described quasi-interpenetrating networks of claim 57 6G/mol.
75., wherein, be limited to 25 * 10 on the weight average molecular weight range of described polydimethylacrylamiin according to the described quasi-interpenetrating networks of claim 57 6G/mol.
76. according to the described quasi-interpenetrating networks of claim 57, wherein, the concentration of described linear polyacrylamide/buffer agent solution is 1.0 to 12.0%g/ml.
77. according to the described quasi-interpenetrating networks of claim 57, wherein, the concentration of described polydimethylacrylamiin/buffer agent solution is 0.1 to 3.0%g/ml.
78. the method for a separating charged minute subclass material, described method comprises: under the effect of extra electric field, charged molecule class material is moved in separating medium, wherein, improvement to described separating medium comprises: linear polyacrylamide polymeric system and polydimethylacrylamiin polymeric system, wherein said polymeric system forms according to each described quasi-interpenetrating networks in the claim 1 to 56.
79. according to the described method of claim 78, wherein, described interpenetrating(polymer)networks be by the main skeleton of synthesizing linear polyacrylamide and in described main skeleton the polymerization DMAA prepare.
80. according to the described method of claim 78, wherein, prepare described interpenetrating(polymer)networks and be by:
(a) provide a kind of solution that comprises linear polyacrylamide and buffer reagent, wherein, the weight-average molecular weight of described linear polyacrylamide is 0.05 * 10 6To 25 * 10 6G/mol;
(b) provide a kind of solution that comprises polydimethylacrylamiin and buffer reagent, wherein, the weight-average molecular weight of described polydimethylacrylamiin is 100000 to 25 * 10 6G/mol; And
(c) in the substep mode linear polyacrylamide/buffer agent solution and polydimethylacrylamiin/buffer agent solution are mixed, wherein, the concentration of described linear polyacrylamide/buffer agent solution is 1 to 15 times of polydimethylacrylamiin/buffer agent solution concentration, and the volume of described linear polyacrylamide/buffer agent solution is 1 to 50 times of polydimethylacrylamiin liquor capacity.
81. the quasi-interpenetrating networks of a polymer chain, described polymer chain comprises:
(a) acrylamide that exists with main skeleton form/DMAA random copolymers chain, the weight-average molecular weight of wherein said random copolymers chain is 0.05 * 10 6To 2 * 10 6G/mol, rotation radius is that 10nm is to 80nm; And
(b) polydimethylacrylamiin chain, wherein said polydimethylacrylamiin chain are in the presence of the main skeleton of random copolymers, prepare by the polymerization DMAA;
Wherein, described acrylamide/DMAA chain and polydimethylacrylamiin chain tangle each other, IPN each other, and wherein said quasi-interpenetrating networks does not have chemically crosslinked.
82. 1 described network according to Claim 8, wherein, the main skeleton of random copolymers forms by radical polymerization.
83. 1 described network according to Claim 8, wherein, described polydimethylacrylamiin is that polymerization obtains by radical polymerization in the main skeleton of random copolymers.
84. 1 described network according to Claim 8, wherein, the main skeleton of described random copolymers comprises the multipolymer in the buffered soln, and the overlapping concentration of wherein said random copolymers is 3 * 10 -3To 4.0 * 10 -2G/ml.
85. 1 described network according to Claim 8, wherein, the overlapping concentration of the main skeleton of described multipolymer in buffered soln is 1 * 10 -2G/ml.
86. 1 described network according to Claim 8, wherein, the following of the weight average molecular weight range of described random copolymers is limited to 0.05 * 10 6G/mol.
87. 1 described network according to Claim 8, wherein, the following of the weight average molecular weight range of described random copolymers is limited to 0.1 * 10 6G/mol.
88. 1 described network according to Claim 8, wherein, the following of the weight average molecular weight range of described random copolymers is limited to 0.3 * 10 6G/mol.
89. 1 described network wherein, is limited to 0.5 * 10 on the weight average molecular weight range of described random copolymers according to Claim 8 6G/mol.
90. 1 described network wherein, is limited to 1 * 10 on the weight average molecular weight range of described random copolymers according to Claim 8 6G/mol.
91. 1 described network wherein, is limited to 2 * 10 on the weight average molecular weight range of described random copolymers according to Claim 8 6G/mol.
92. 1 described network according to Claim 8, wherein, the following 10nm that is limited to of the rotation radius scope of described multipolymer.
93. 1 described network according to Claim 8, wherein, the following 15nm that is limited to of the rotation radius scope of described multipolymer.
94. 1 described network according to Claim 8, wherein, the following 30nm that is limited to of the rotation radius scope of described multipolymer.
95. 1 described network wherein, is limited to 40nm on the rotation radius scope of described multipolymer according to Claim 8.
96. 1 described network wherein, is limited to 55nm on the rotation radius scope of described multipolymer according to Claim 8.
97. 1 described network wherein, is limited to 80nm on the rotation radius scope of described multipolymer according to Claim 8.
98. 1 described network according to Claim 8, wherein, the polydispersity index of described random copolymers is 1.1 to 2.0.
99. the quasi-interpenetrating networks of the polymer chain of an entanglement, described quasi-interpenetrating networks is prepared by following method, and described method comprises:
(a) provide a kind of solution that comprises acrylamide/DMAA random copolymers and buffer reagent, wherein, the weight-average molecular weight of described acrylamide/DMAA random copolymers is 0.05 * 10 6To 2 * 10 6G/mol;
(b) provide a kind of solution that comprises polydimethylacrylamiin and buffer reagent, wherein, the weight-average molecular weight of described polydimethylacrylamiin is 0.05 * 10 6To 25 * 10 6G/mol;
(c) in the substep mode multipolymer/buffer agent solution and polydimethylacrylamiin/buffer agent solution are mixed, wherein, the concentration of described multipolymer/buffer agent solution is 1 to 50 times of polydimethylacrylamiin/buffer agent solution concentration, and the volume of described multipolymer/buffer agent solution is 1 to 20 times of polydimethylacrylamiin/buffer agent solution volume;
Wherein, prepare the copolymer chain of entanglement and the quasi-interpenetrating networks of polydimethylacrylamiin polymer chain, wherein, described quasi-interpenetrating networks does not have chemically crosslinked.
100. according to the described quasi-interpenetrating networks of claim 99, wherein, the following of the weight average molecular weight range of described multipolymer is limited to 0.05 * 10 6G/mol.
101. according to the described quasi-interpenetrating networks of claim 99, wherein, the following of the weight average molecular weight range of described multipolymer is limited to 0.1 * 10 6G/mol.
102. according to the described quasi-interpenetrating networks of claim 99, wherein, the following of the weight average molecular weight range of described multipolymer is limited to 0.3 * 10 6G/mol.
103., wherein, be limited to 0.5 * 10 on the weight average molecular weight range of described multipolymer according to the described quasi-interpenetrating networks of claim 99 6G/mol.
104., wherein, be limited to 1 * 10 on the weight average molecular weight range of described multipolymer according to the described quasi-interpenetrating networks of claim 99 6G/mol.
105., wherein, be limited to 1.5 * 10 on the weight average molecular weight range of described multipolymer according to the described quasi-interpenetrating networks of claim 99 6G/mol.
106., wherein, be limited to 2 * 10 on the weight average molecular weight range of described multipolymer according to the described quasi-interpenetrating networks of claim 99 6G/mol.
107. according to the described quasi-interpenetrating networks of claim 99, wherein, the following 50000g/mol that is limited to of the weight average molecular weight range of described polydimethylacrylamiin.
108. according to the described quasi-interpenetrating networks of claim 99, wherein, the following 100000g/mol that is limited to of the weight average molecular weight range of described polydimethylacrylamiin.
109. according to the described quasi-interpenetrating networks of claim 99, wherein, the following 200000g/mol that is limited to of the weight average molecular weight range of described polydimethylacrylamiin.
110., wherein, be limited to 500000g/mol on the weight average molecular weight range of described polydimethylacrylamiin according to the described quasi-interpenetrating networks of claim 99.
111., wherein, be limited to 1 * 10 on the weight average molecular weight range of described polydimethylacrylamiin according to the described quasi-interpenetrating networks of claim 99 6G/mol.
112., wherein, be limited to 3 * 10 on the weight average molecular weight range of described polydimethylacrylamiin according to the described quasi-interpenetrating networks of claim 99 6G/mol.
113., wherein, be limited to 5 * 10 on the weight average molecular weight range of described polydimethylacrylamiin according to the described quasi-interpenetrating networks of claim 99 6G/mol.
114., wherein, be limited to 10 * 10 on the weight average molecular weight range of described polydimethylacrylamiin according to the described quasi-interpenetrating networks of claim 99 6G/mol.
115., wherein, be limited to 25 * 10 on the weight average molecular weight range of described polydimethylacrylamiin according to the described quasi-interpenetrating networks of claim 99 6G/mol.
116. according to the described quasi-interpenetrating networks of claim 99, wherein, the concentration of described multipolymer/buffer agent solution is 5.0 to 20.0%g/ml.
117. according to the described quasi-interpenetrating networks of claim 99, wherein, the concentration of described polydimethylacrylamiin/buffer agent solution is 0.1 to 1.0%g/ml.
118. the method for a separating charged minute subclass material, described method comprises: under the influence of extra electric field, charged molecule class material is moved in separating medium, wherein the improvement to described separating medium comprises: acrylamide/DMAA random copolymers and polydimethylacrylamiin polymkeric substance, wherein, described polymeric system forms according to Claim 8 each described quasi-interpenetrating networks in 1 to 98.
119. according to the described method of claim 118, wherein, described interpenetrating(polymer)networks be by synthesis of acrylamide/main skeleton of DMAA random copolymers and in described main skeleton the polymerization DMAA prepare.
120. according to the described method of claim 118, wherein, prepare described interpenetrating(polymer)networks and be by:
(a) provide a kind of solution that comprises acrylamide/DMAA random copolymers and buffer reagent, wherein, the weight-average molecular weight of acrylamide/DMAA random copolymers is 0.05 * 10 6To 2 * 10 6G/mol;
(b) provide a kind of solution that comprises polydimethylacrylamiin and buffer reagent, wherein, the weight-average molecular weight of polydimethylacrylamiin is 50,000 to 25 * 10 6G/mol; And
(c) with the substep form multipolymer/buffer agent solution and polydimethylacrylamiin/buffer agent solution are mixed, the concentration of wherein said multipolymer/buffer agent solution is 1 to 50 times of polydimethylacrylamiin/buffer agent solution concentration, and the volume of described multipolymer/buffer agent solution is 1 to 20 times of polydimethylacrylamiin liquor capacity;
Wherein, prepare the multipolymer of entanglement and the quasi-interpenetrating networks of polydimethylacrylamiin polymer chain, wherein, described quasi-interpenetrating networks does not have chemically crosslinked.
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