CN1966719B - Polymer/gold nano particle composite medium for use in capillary electrophoresis DNA sequencing and method for preparing same - Google Patents

Polymer/gold nano particle composite medium for use in capillary electrophoresis DNA sequencing and method for preparing same Download PDF

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CN1966719B
CN1966719B CN2006101140993A CN200610114099A CN1966719B CN 1966719 B CN1966719 B CN 1966719B CN 2006101140993 A CN2006101140993 A CN 2006101140993A CN 200610114099 A CN200610114099 A CN 200610114099A CN 1966719 B CN1966719 B CN 1966719B
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dna sequencing
accurate
nano particle
gold nano
capillary electrophoresis
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CN1966719A (en
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王延梅
周丹
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University of Science and Technology of China USTC
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Abstract

The invention relates to polymers/gold nanoparticles complex medium for DNA sequencing with capillary electrophoresis and its preparation method, and belongs to the biotechnological field of DNA sequencing and separation. The concentration of the buffer solution of polymers/gold nanoparticles complex medium used for DNA sequencing is 2.0%-3.0%. The invention comprises the steps of: (1) synthesis of quasi-interpenetrating polymer networks with self-spreading ability, (2) preparation of Au nanoparticles, and (3) synthesis of complex medium of quasi-interpenetrating polymer networks/gold nanoparticles. The complex medium with reduced viscosity at proper concentration is easy to be pumped into and run out of the capillaries, and can prevent the production of electroosmotic flow because of the existence of complex with self spreading ability, which saves the step of spreading inner wall of the capillary, simplifies the preparation process of column, reduces the application cost of capillary, elongates the service time of capillary. The invention is mainly used for separating DNA with capillary electrophoresis and DNA sequencing.

Description

A kind of polymer/gold nano particle composite medium that is used for capillary electrophoresis DNA sequencing and preparation method thereof
Technical field
The present invention relates to a kind of polymer/gold nano particle composite medium that is used for capillary electrophoresis DNA sequencing and preparation method thereof, belong to dna sequencing and separating bio technical field.
Background technology
Capillary electrophoresis is to carry out the DNA analysis efficient ways.For capillary electrophoresis, because separating medium is determining the migratory behaviour and the resolution of DNA fragment, thereby it is the isolating important factor of DNA, synthetic macromolecule is the separating medium of using during DNA separates the most widely at present, for an effective separating medium, should possess high sieving capacity, low viscosity simultaneously and apply function certainly.
The synthetic polymer that is usually used in DNA isolation mainly contains hydrophilic homopolymer (Heller C., Electrophoresis, 2001,22:629-643), concerning these homopolymer, major part need apply to remove electroosmotic flow capillary tube inner wall, wherein has only PEO (polyethylene oxide), PDMA (poly-N,N-DMAA), PVP minority polymkeric substance such as (Polyvinylpyrolidone (PVP)s) that the function from coating is arranged.Comparatively speaking, the separating effect of high molecular weight linear polyacrylamide (LPA) is best in the homopolymer at present, but it does not apply function certainly, and viscosity is also higher under the concentration of dna sequencing, therefore causes adorning automatically the post difficulty.In order to address this problem, (Wang Y, Liang D, Ying Q such as wang, .Chu B, Electrophoresis, 2005,26:126-136) aforesaid method is improved, initiated polymerization DMA (N in the high-molecular weight LPA aqueous solution, the N-DMAA) obtains accurate interpenetrating polymer networks (quasi-IPN), regulate AA (acrylamide) and find, providing under enough coating ability conditions the few more separation efficiency of DMA content good more with the ratio of DMA.But the molecular weight (～10 of LPA in the employed medium of this method 7) too high, when being dissolved in the buffered soln with certain concentration, high-molecular weight LPA degrade easily its work-ing life (≤4 months) be subjected to very big influence, in addition, because molecular weight is too high, causes viscosity too high, therefore need to use the high-pressure system injection or go out kapillary, increase analysis cost on the one hand, be difficult to realize the automatic exchange of sieving medium on the other hand.Have higher sieving capacity and read the common viscosity of macromolecular solution of length than length dna also higher, therefore, the low viscosity sieving medium of seeking to have high sieving capacity is still the important topic of DNA high efficiency separation.
In recent years, adding various additives (as polyol, clay, golden nanometer particle, emulsion particle etc.) verified in the low viscosity macromolecular solution is very effective and simple method, sieving capacity when sieving medium in the presence of the additive can just reach high density (additive-free) under low concentration becomes the effective way that does not increase viscosity and improve separating power.People such as Huang (Huang MF, Huang CC, Chang HT.Electrophoresis, 2003,24:2896-2902) propose to separate dsDNA (double-stranded DNA) fragment as separating medium with the PEO solution that contains GNPs (golden nanometer particle), for fear of the interaction of capillary wall and DNA, kapillary has been carried out dynamic coating with 5.0%PVP.Since viscosity pole low (＜15cP), be easy to carry out the automatic replacing of sieving medium, studies show that the PEO that contains GNPs is used for having the following advantages when dsDNA separates: fast, high, the favorable reproducibility of separating power and be easy to filled capillary pipe.But also because viscosity pole is low, this medium can not sort to DNA.
Summary of the invention
The present invention introduces golden nanometer particle in this polymkeric substance then in conjunction with inverse emulsion polymerization and the synthetic accurate interpenetrating polymer networks that has the hydrophilic polymer of separation function and apply the polymkeric substance composition of function certainly of solution polymerization process.
The purpose of this invention is to provide a kind of polymer/gold nano particle composite medium that is used for capillary electrophoresis DNA sequencing, this kind complex media is in empty kapillary, under the lower viscosity, can sort to the DNA base, prolong the work-ing life that is dissolved in the complex media in the buffered soln, save this step of coating capillary tube inner wall, simplified system post process greatly; Golden nanometer particle can form the physical crosslinking point in accurate interpenetrating polymer networks, strengthened the validity of the polymer network that forms in the sepn process, remedied the not too high defective of separating medium polymericular weight, thereby make the separating medium that obtains reach low viscosity, have from applying function, can form polymer network efficiently again, improve the speed and the efficient of DNA separation and dna sequencing.
Another object of the present invention provides a kind of preparation method who is used for the polymer/gold nano particle composite medium of capillary electrophoresis DNA sequencing.
Realize technical scheme of the present invention:
The invention provides a kind of polymer/gold nano particle composite medium that is used for capillary electrophoresis DNA sequencing, combine, it is characterized in that the viscosity-average molecular weight of linear polyacrylamide: 0.9 * 10 by accurate interpenetrating polymer networks and golden nanometer particle 6～3.5 * 10 6Da, acrylamide: N, N-DMAA=10: 1～100: 1 (mol ratio), the diameter of Au is 10～65nm, Au content is 20～1200 μ g in every gram polymer/gold nano particle composite medium, the buffer concentration that is used for the polymer/gold nano particle composite medium of dna sequencing is 2.0%～3.0%, and wherein the viscosity-average molecular weight of preferred linear polyacrylamide is 3.0 * 10 6Da.
This complex media is prepared from by following method:
(1) have from applying the synthetic of the accurate interpenetrating polymer networks of function:
The method of at first using reverse emulsion polymerization is in kerosene, Span-80, aqueous systems, obtain linear polyacrylamide with ammonium persulphate/Tetramethyl Ethylene Diamine is acrylamide triggered, the shared mass percent of each component is in whole reaction system: Span-80 is 2～3%, kerosene is 37～43%, water is 31～37%, acrylamide is 20～26%, and ammonium persulphate is 0.002～0.008%, and Tetramethyl Ethylene Diamine is 0.002～0.005%; Concrete reactions steps is: Span-80 is mixed with kerosene, after stirring, logical nitrogen adds the solution that acrylamide and water are formed, logical nitrogen 1 hour, under 20 ℃～40 ℃ temperature, after adding the aqueous solution and Tetramethyl Ethylene Diamine of 10% (w/v) ammonium persulphate, reacted 16～30 hours, with products therefrom with acetone immersion, suction filtration, washing, for several times, obtain the linear polyacrylamide of product continuously;
1% (w/v) polyacrylamide solution under stirring, the speed of 100rpm is fed nitrogen after 0.5～2 hour, add N, the N-DMAA, wherein linear polyacrylamide and N, the mass ratio of N-DMAA is 1: 1～5: 1, logical nitrogen is after 5～30 minutes, the ammonium persulfate aqueous solution that adds 10% (w/v), N wherein, the mass ratio of N-DMAA and ammonium persulphate is 1: 0.01～1: 0.04, and the mass ratio of ammonium persulphate and Tetramethyl Ethylene Diamine is 1: 2～2: 1, and-2～2 ℃ were reacted 16～30 hours down, products therefrom is soaked with acetone, suction filtration, washing, continuously for several times;
(2) preparation of Au nanoparticle:
Trisodium Citrate reduction HAuCl with routine 4The aqueous solution makes Au colloidal sol;
(3) accurate interpenetrating polymer networks/gold nano particle composite medium is synthetic:
With above-mentioned accurate interpenetrating polymer networks and Au colloidal sol according to accurate interpenetrating polymer networks: the mixed of Au colloidal sol=1g: 0.5～25mL, use acetone precipitation, filter, drying, obtain accurate interpenetrating polymer networks/gold nano particle composite medium.
The more concrete preparation method of this complex media is:
(1) has from applying the synthetic of the accurate interpenetrating polymer networks of function (quasi-IPN)
Accurate interpenetrating polymer networks is the polymer network that two kinds of polymkeric substance of non-crosslinked run through composition mutually, the polymer network that interweaves that forms and interpenetrating polymer networks is meant that two or more cross-linked polymer runs through mutually.
The method of at first using inverse emulsion polymerization obtains linear polyacrylamide (LPA) with ammonium persulphate (APS)/Tetramethyl Ethylene Diamine (TEMED) acrylamide triggered (AM) in kerosene, Span-80, aqueous systems.The shared mass percent of each component in whole reaction system: water is 31～37%, kerosene is 37～43%, AM is 20～26%, Span-80 is 2～3%, APS is 0.002～0.008%, and TEMED is 0.002～0.005%.Concrete reactions steps is as follows: measure quantitative Span-80 and be dissolved in an amount of kerosene, logical nitrogen deoxygenation, mechanical stirring 500rpm.Take by weighing quantitative acrylamide and be dissolved in appropriate amount of deionized water, obtain colourless transparent solution after treating to dissolve fully, dropwise splash in the four-necked bottle with dropping funnel, system presents the emulsion state at first, becomes oyster white subsequently gradually.Continuously logical nitrogen deoxygenation heated up after 1 hour, added an amount of APS aqueous solution and TEMED respectively with micro-syringe, reacted 16～30 hours.Question response under agitation splashes into reactant a large amount of acetone after finishing, and obtains white precipitate, vacuum-drying under the room temperature, then that product is soluble in water again, with acetone precipitation, suction filtration, vacuum-drying under the product room temperature that obtains, so triplicate obtains the product polyacrylamide, is determined intrinsic viscosity [η] (the 0.1mol/L NaCl aqueous solution of product by Ubbelohde viscometer, 30 ℃), pass through the viscosity-average molecular weight M that the Mark-Houwink formula calculates product again v:
[η]＝9.33×10 -3M v 0.75(mL/g)
Add in the aqueous solution of polyacrylamide then and have from the monomer that applies function (as DMA etc.), the mode that causes with oxidation-reduction is carried out polymerization and is obtained having accurate interpenetrating polymer networks from applying function.Concrete reactions steps is as follows: add polyacrylamide solution in four-necked bottle, mechanical stirring 50rpm feeds nitrogen and removes oxygen in the system.Deoxygenation added an amount of N,N-DMAA with syringe after 1 hour, continued logical nitrogen and added an amount of the APS aqueous solution and TEMED with micro-syringe after ten minutes, 0 ℃ of reaction 24 hours down.Question response under agitation splashes into reactant a large amount of acetone after finishing, and obtains white precipitate, with acetone immersion, suction filtration, washing, for several times, the solid that obtains is placed vacuum drying oven continuously, and vacuum-drying is to constant weight under the room temperature.The proportioning of AM and DMA by proton nmr spectra ( 1H-NMR) determine;
(2) preparation of Au nanoparticle
Preparing required glassware all needs to clean with chloroazotic acid, then with deionized water rinsing and dry.In being housed, the round-bottomed flask of prolong adds an amount of HAuCl 4The aqueous solution, this solution of heating is to boiling under vigorous stirring.Fast an amount of sodium citrate aqueous solution is joined in the above-mentioned boiling solution, this solution colour from the faint yellow blueness that becomes, becomes red-purple rapidly then, shows to have generated golden nanometer particle (GNPs).After continuing boiling 10min, remove thermal source.Colloidal sol then stirs the 15min postcooling to room temperature.The big I of GNPs records by transmission electron microscope.
(3) accurate interpenetrating polymer networks/gold nano particle composite medium is synthetic
Above-mentioned Au colloidal sol is added respectively in an amount of quasi-IPN aqueous solution.After mixing fully, precipitate this solution, filter and vacuum-drying, obtain polymer/gold nano particle composite medium with excessive propanone.
Reaction process is as follows:
Beneficial effect of the present invention: the present invention in conjunction with the synthetic hydrophilic polymer with separation function of inverse emulsion polymerization and solution polymerization process (as polyacrylamide etc.) and the polymkeric substance that applies function certainly (as poly-N, N-DMAA etc.) the accurate interpenetrating polymer networks of Zu Chenging, then golden nanometer particle is introduced in this polymer network, formed polymer/gold nano particle composite medium.It is the accurate interpenetrating(polymer)networks of physical crosslinking point that this medium can form with the golden nanometer particle under certain concentration in buffered soln, with this network during as the sieving medium of no glue capillary electrophoresis, because its major portion of parting material is the polyacrylamide with separation function, and when the molecular weight of polyacrylamide is very big (～10 7), reduce separation efficiency thereby in buffered soln, degrade easily, so this project is because isolating polymer (LPA) molecular weight (～10 that uses 6) not very high, therefore under dna sequencing concentration (2.0%～3.0%), viscosity not high (intrinsic viscosity of this medium [η] ≈ 700mL/g) can be used commercial apparatus ABI-310 automatic sequencing; And in the accurate interpenetrating(polymer)networks that LPA and PDMA form in background technology, when the molecular weight of LPA near 10 7The time, under dna sequencing concentration (2.0%～2.5%), viscosity higher (intrinsic viscosity of this medium [η] ≈ 1500mL/g) owing to need the high-pressure system sample introduction, therefore is difficult to adorn automatically post.Separating medium viscosity under certain concentration is reduced, be convenient to inject and go out kapillary, reduce the possibility of polymer degradation in the preservation process of separating medium solution on the other hand; Because the existence from applying functional polymer need not to apply in advance capillary tube inner wall, just can prevent the generation of electroosmotic flow, save this step of coating capillary tube inner wall, simplify system post process greatly, reduced use cost capillaceous, increased work-ing life capillaceous; Because golden nanometer particle can become the physical crosslinking point of network in accurate interpenetrating(polymer)networks, strengthened the validity of the polymer network that forms in the sepn process like this, remedied the defective that the not too high efficient of bringing of main polymericular weight reduces in the parting material, prolonged the shelf-time of separating medium solution, this separating medium shelf-time in buffered soln reached more than 8 months, and high molecular (～10 7) the shelf-time of polymkeric substance in buffered soln less than 4 months.Be 6 months the work-ing life of general commercial medium; Simultaneously with background technology in the PEO/Au compound system can only separate the dsDNA fragment and compare, this separating medium can sort to ssDNA under certain concentration (2.0%～3.0%).Can obtain low viscosity, have by this project, can fundamentally be familiar with DNA separation and the speed of order-checking and the relation between the efficient by the research of separating mechanism from applying function and the high separating medium of efficient.
Description of drawings
Fig. 1: the composition principle figure that is polymer/gold nano particle composite medium;
Fig. 2: the 1 * TTE/7M urea soln of quasi-IPN/GNPs-1 that is 2.5% (w/v) is to the sequencer map of standard model DNA (Bigdye Terminator V 3.1 sequencing standard) base A;
Fig. 3: the 1 * TTE/7M urea soln of quasi-IPN/GNPs-1 that is 2.5% (w/v) is to the part sequencer map of four bases of standard model DNA (Bigdye Terminator V 3.1 sequencing standard);
Fig. 4: the 1 * TTE/7M urea soln of quasi-IPN/GNPs-2 that is 2.5% (w/v) is to the sequencer map of standard model DNA (Bigdye Terminator V 3.1 sequencing standard) base A;
Fig. 5: the 1 * TTE/7M urea soln of quasi-IPN/GNPs-3 that is 2.5% (w/v) is to the sequencer map of standard model DNA (Bigdye Terminator V 3.1 sequencing standard) base A;
Fig. 6: the 1 * TTE/7M urea soln of quasi-IPN/GNPs-4 that is 3.0% (w/v) is to the sequencer map of standard model DNA (Bigdye Terminator V 3.1 sequencing standard) base A;
Fig. 7: the 1 * TTE/7M urea soln of quasi-IPN/GNPs-5 that is 2.5% (w/v) is to the sequencer map of standard model DNA (Bigdye Terminator V 3.1 sequencingstandard) base A;
Fig. 8: the 1 * TTE/7M urea soln of quasi-IPN/GNPs-6 that is 2.0% (w/v) is to the sequencer map of standard model DNA (Bigdye Terminator V 3.1 sequencing standard) base A.
Followingly in conjunction with the embodiments the present invention is done further qualification, but the present invention is not limited only to following examples.
Embodiment
Embodiment 1
(1) preparation of the accurate interpenetrating polymer networks of LPA/PDMA
With Span 80 (2.47g), refined kerosene (50mL) joins in the four neck round-bottomed flasks of 250mL clean dried, and installs mechanical stirrer, reflux condensing tube, nitrogen importing/delivery line and constant pressure funnel on flask.With acrylamide (AM, 20g) and H 2The solution of O (30g) dropwise adds in the mixture.At the mechanical stirring rotating speed is under the 500rpm condition, adds 40 μ L APS (0.1g/mL) aqueous solution and 4.5 μ L TEMED behind feeding ultra-pure nitrogen (UHP, 99.99%) 1h continuously in this system, reacts 24h down at 22 ℃.After polymerization finishes, emulsion is precipitated out in excessive propanone, filters, water dissolution is used in vacuum-drying then, uses acetone precipitation again, and vacuum-drying three times like this, obtains the linear polyacrylamide (LPA) of purifying.The viscosity-average molecular weight that records LPA with dark type viscometer is 3.0 * 10 6Da.
In the three-necked bottle of 100mL clean dried, add 50mL 1% (w/v) the LPA aqueous solution; logical UHP nitrogen; mechanical stirring; rotating speed is 100rpm; 0 ℃ adds down 0.2mL DMA, adds 50 μ L APS (0.1g/mL) aqueous solution and 8 μ L TEMED behind the 1h in this system, behind the 0.5h stirring velocity is adjusted to 50rpm; under nitrogen protection, continue reaction 24h.Final product precipitates with excessive propanone, vacuum-drying then, 1H-NMR result shows, AM: DMA=30: 1.
(2) preparation of 40nm Au particle
Required glassware all will clean with chloroazotic acid, then with deionized water rinsing and dry.In being housed, the round-bottomed flask of prolong adds 50mL HAuCl 4(0.01%, the wt.) aqueous solution, this solution of heating is to boiling under intense agitation.(1%, wt.) aqueous solution joins in the above-mentioned boiling solution, and this solution colour from the faint yellow blueness that becomes, becomes red-purple rapidly then, continues to seethe with excitement behind the 10min, removes thermal source with the 0.5mL Trisodium Citrate fast.Colloidal sol then stirs the 15min postcooling to room temperature.Transmission electron microscope results shows that the mean diameter of Au is 40nm.
(3) preparation of sieving medium quasi-IPN/GNPs-1 and preparation
The above-mentioned Au colloidal sol of 0.25mL is added to respectively in the above-mentioned quasi-IPN aqueous solution of 20mL 1% (w/v).After mixing fully, precipitate this solution, filter and vacuum-drying with excessive propanone.
When carrying out the dna sequencing experiment, this medium dissolves with 1 * TTE (50mM Tris (Tutofusin tris)/50mM TAPS (N-three (methylol) methyl-3-amino propane sulfonic acid)/2.0mM EDTA (ethylenediamine tetraacetic acid (EDTA))) buffered soln, and is diluted to 2.5% (the macromolecular solution concentration unit is w/v).
The implementation condition of capillary electrophoresis is as follows:
Empty kapillary useful length/length overall: 50/61cm; Capillary inner diameter/external diameter: 75/365u m; DNA sample introduction voltage is 2.5KV, and sample injection time is 30s; Order-checking voltage: 9.1KV; The order-checking temperature is 50 ℃; The used damping fluid of anode is 1 * TTE in the dna sequencing process, and the used damping fluid of negative electrode is 1 * TTE/7M urea.During experiment earlier with deionized water with empty capillary douche 60 seconds.
The dna sequencing spectrogram that 2.5% quasi-IPN/GNPs-1 obtains as sieving medium is as Fig. 2, shown in Figure 3.
Embodiment 2
(1) preparation of the accurate interpenetrating(polymer)networks of LPA/PDMA
Method is with embodiment one.
(2) preparation of 40nmAu particle
Method is with embodiment one.
Au particle diameter identical and preparation and the preparation of the sieving medium quasi-IPN/GNPs-2 that content is different
(3) preparation of sieving medium quasi-IPN/GNPs-1 and preparation
The above-mentioned Au colloidal sol of 2.0mL is added to respectively in the above-mentioned quasi-IPN aqueous solution of 20mL 1% (w/v).After mixing fully, precipitate this solution, filter and vacuum-drying with excessive propanone.
When carrying out the dna sequencing experiment, this medium dissolves with 1 * TTE buffered soln, and is diluted to 2.5% (the macromolecular solution concentration unit is w/v).
The implementation condition of capillary electrophoresis is with embodiment one.
The dna sequencing spectrogram that 2.5% quasi-IPN/GNPs-2 obtains as sieving medium, as shown in Figure 4.
Embodiment 3
(1) preparation of the accurate interpenetrating(polymer)networks of LPA/PDMA
Method is with embodiment one.
(2) preparation of 20nmAu particle
Required glassware all will clean with chloroazotic acid, then with deionized water rinsing and dry.In being housed, the round-bottomed flask of prolong adds 50mL 0.01% (wt.) HAuCl 4The aqueous solution, this solution of heating is to boiling under intense agitation.Fast 1% (wt.) sodium citrate aqueous solution 0.85mL is joined in the above-mentioned boiling solution, this solution colour from the faint yellow blueness that becomes, becomes red-purple rapidly then, continues to seethe with excitement behind the 10min, removes thermal source.Colloidal sol then stirs the 15min postcooling to room temperature.Transmission electron microscope results shows that the mean diameter of Au is 20nm.
(3) the Au particle diameter is preparation and the preparation of the sieving medium quasi-IPN/GNPs-3 of 20nm
The above-mentioned Au colloidal sol of 1.0mL is added to respectively in the above-mentioned quasi-IPN aqueous solution of 20mL 1% (w/v).After mixing fully, precipitate this solution, filter and vacuum-drying with excessive propanone.
When carrying out the dna sequencing experiment, this medium dissolves with 1 * TTE buffered soln, and is diluted to 2.5% (the macromolecular solution concentration unit is w/v).
The implementation condition of capillary electrophoresis is with embodiment one.
The dna sequencing spectrogram that 2.5% quasi-IPN/GNPs-3 obtains as sieving medium, as shown in Figure 5.
Embodiment 4
(1) preparation of the accurate interpenetrating(polymer)networks of LPA/PDMA
With Span 80 (2.47g), refined kerosene (50mL) joins in the four neck round-bottomed flasks of 250mL clean dried, and installs mechanical stirrer, reflux condensing tube, nitrogen importing/delivery line and constant pressure funnel on flask.With acrylamide (AM, 20g) and H 2The solution of O (30g) dropwise adds in the mixture.At the mechanical stirring rotating speed is under the 500rpm condition, adds 80 μ L APS (0.1g/mL) aqueous solution and 4.5 μ L TEMED behind feeding ultra-pure nitrogen (UHP, 99.99%) 1h continuously in this system, reacts 24h down at 40 ℃.After polymerization finishes, emulsion is precipitated out in excessive propanone, filters, water dissolution is used in vacuum-drying then, uses acetone precipitation again, and vacuum-drying three times like this, obtains the LPA of purifying.The viscosity-average molecular weight that records LPA with dark type viscometer is 0.93 * 10 6Da.
In the three-necked bottle of 100mL clean dried, add 50mL 1% (w/v) the LPA aqueous solution; logical UHP nitrogen; mechanical stirring; rotating speed is 100rpm; 0 ℃ adds down 0.30mL DMA, adds 100 μ L APS (0.1g/mL) aqueous solution and 8 μ L TEMED behind the 1h in this system, behind the 0.5h stirring velocity is adjusted to 50rpm; under nitrogen protection, continue reaction 24h.Final product precipitates with excessive propanone, vacuum-drying then, 1H-NMR result shows, AM: DMA=10: 1.
(2) preparation of 40nm Au particle
Method is with embodiment one.
(3) the Au particle diameter is preparation and the preparation of the sieving medium quasi-IPN/GNPs-4 of 40nm
The above-mentioned Au colloidal sol of 2.0mL is added to respectively in the above-mentioned quasi-IPN aqueous solution of 20mL 1% (w/v).After mixing fully, precipitate this solution, filter and vacuum-drying with excessive propanone.
When carrying out the dna sequencing experiment, this medium dissolves with 1 * TTE buffered soln, and is diluted to 3.0% (the macromolecular solution concentration unit is w/v).
The implementation condition of capillary electrophoresis is with embodiment one.
The dna sequencing spectrogram that 3.0% quasi-IPN/GNPs-4 obtains as sieving medium, as shown in Figure 6.
Embodiment 5
(1) preparation of the accurate interpenetrating(polymer)networks of LPA/PDMA
With Span 80 (2.47g), refined kerosene (50mL) joins in the four neck round-bottomed flasks of 250mL clean dried, and installs mechanical stirrer, reflux condensing tube, nitrogen importing/delivery line and constant pressure funnel on flask.With acrylamide (AM, 20g) and H 2The solution of O (30g) dropwise adds in the mixture.At the mechanical stirring rotating speed is under the 500rpm condition, adds 80 μ L APS (0.1g/mL) aqueous solution and 4.5 μ L TEMED behind feeding ultra-pure nitrogen (UHP, 99.99%) 1h continuously in this system, reacts 24h down at 40 ℃.After polymerization finishes, emulsion is precipitated out in excessive propanone, filters, water dissolution is used in vacuum-drying then, uses acetone precipitation again, and vacuum-drying three times like this, obtains the LPA of purifying.The viscosity-average molecular weight that records LPA with dark type viscometer is 0.93 * 10 6Da.
In the three-necked bottle of 100mL clean dried, add 50mL 1% (w/v) the LPA aqueous solution; logical UHP nitrogen; mechanical stirring; rotating speed is 100rpm; 0 ℃ adds down 0.30mL DMA, adds 15 μ LAPS (0.1g/mL) aqueous solution and 8 μ L TEMED behind the 1h in this system, behind the 0.5h stirring velocity is adjusted to 50rpm; under nitrogen protection, continue reaction 24h.Final product precipitates with excessive propanone, vacuum-drying then, 1H-NMR result shows, AM: DMA=100: 1.
(2) preparation of 65nm Au particle
Required glassware all will clean with chloroazotic acid, then with deionized water rinsing and dry.In being housed, the round-bottomed flask of prolong adds 50mL 0.01% (wt.) HAuCl 4The aqueous solution, this solution of heating is to boiling under intense agitation.Fast 1% (wt.) sodium citrate aqueous solution 0.35mL is joined in the above-mentioned boiling solution, continue to seethe with excitement behind the 15min, remove thermal source.Colloidal sol then stirs the 15min postcooling to room temperature.Transmission electron microscope results shows that the mean diameter of Au is 65nm.
(3) the Au particle diameter is preparation and the preparation of the sieving medium quasi-IPN/GNPs-5 of 65nm
The above-mentioned Au colloidal sol of 1.0mL is added to respectively in the above-mentioned quasi-IPN aqueous solution of 20mL 1% (w/v).After mixing fully, precipitate this solution, filter and vacuum-drying with excessive propanone.
When carrying out the dna sequencing experiment, this medium dissolves with 1 * TTE buffered soln, and is diluted to 2.5% (the macromolecular solution concentration unit is w/v).
The implementation condition of capillary electrophoresis is with embodiment one.
The dna sequencing spectrogram that 2.5% quasi-IPN/GNPs-5 obtains as sieving medium, as shown in Figure 7.
Embodiment 6
(1) preparation of the accurate interpenetrating(polymer)networks of LPA/PDMA
Method is with embodiment five.
(2) preparation of 65nm Au particle
Method is with embodiment five.
(3) the Au particle diameter is preparation and the preparation of the sieving medium quasi-IPN/GNPs-6 of 65nm
The above-mentioned Au colloidal sol of 2.0mL is added to respectively in the above-mentioned quasi-IPN aqueous solution of 20mL 1% (w/v).After mixing fully, precipitate this solution, filter and vacuum-drying with excessive propanone.
When carrying out the dna sequencing experiment, this medium dissolves with 1 * TTE buffered soln, and is diluted to 2.0% (the macromolecular solution concentration unit is w/v).
The implementation condition of capillary electrophoresis is with embodiment one.
The dna sequencing spectrogram that 2.5% quasi-IPN/GNPs-6 obtains as sieving medium, as shown in Figure 8.
Reagent used among the above embodiment all carries out purifying according to ordinary method.
(1) deionized water (board, China Science ﹠ Technology University are given birth to by section): with the automatic triple pure water distillers of SZ-3 type (Shanghai Hu Xi analytical instrument factory) purifying treatment, final gained electrical conductivity of water is 1.4 * 10 before using -6S/cm.
(2) acetone (China Medicine's analytical pure): add 0.5g potassium permanganate in the 100mL acetone and reflux,, till no longer disappearing to purple, use anhydrous K to remove reducing impurity 2CO 3Perhaps CaCO 3Dry filter is collected 55～56.5 ℃ of cuts.
(3) chloroform: generally contain 1% ethanol, prevent to resolve into phosgene.With the water vibration several of half volume, tell lower floor's chloroform, with distilling behind the dry 12h of Calcium Chloride Powder Anhydrous.Collect 61.5～62 ℃ of cuts.
(4) kerosene (commercially available):, add that with 10% sulfuric acid the saturated solution that potassium permanganate is made into washs again, till the water layer purple no longer disappears with the vitriol oil of its volume 1/10th washing three times.With the washing of sodium hydroxide saturated solution, wash with water at last for several times then,, after the Calcium Chloride Powder Anhydrous drying, distill, collect 180～220 ℃ cut to pH=7.
(5) (love is built Degussa (Shanghai) initiator company limited to ammonium persulphate, analytical pure): in the saturated aqueous solution of 30 ℃ of preparation ammonium persulphates, add a little distilled water after-filtration again and remove insolubles, solution is placed the cooling of the refrigerator degree of depth, separate out the ammonium persulphate crystal.Filter,, use BaCl with the small amount of deionized water washing 2Solution detects whether also have SO in the filtrate 4 2-There is crystallization once more if needed.The vacuum-drying under room temperature of gained crystal, 0 ℃ of airtight preservation down.
(6) acrylamide (China Medicine (Group) Shanghai Chemical Reagent Co.,, analytical pure): the 35g acrylamide, be dissolved in the 500mL chloroform, be heated to 50 ℃, filtered while hot, room temperature is slowly cooled off, and collects crystal.Vacuum-drying 2h under the room temperature.By the same method again recrystallization once, room temperature vacuum-drying 24h to twice weighing 0.001g that is no more than of poor quality, stores in lucifuge kept dry in the brown bottle.
(7) N,N-DMAA (DMA, Aldrich, 〉=99%): underpressure distillation obtains colourless transparent liquid, the lucifuge cryopreservation.
(8) Tutofusin tris (Tris (hydroxylmethyl) aminomethane, Tris), N-three (methylol) methyl-3-amino propane sulfonic acid (N-tris (hydroxylmethyl) methyl-3-aminopropane sulfonic acid, TAPS), ethylenediamine tetraacetic acid (EDTA) (ethylenediaminetetraacetic acid, EDTA), urea, analytical pure is all purchased in Aldrich.
(9) HAuCl 44H 2O (China Medicine (Group) Shanghai Chemical Reagent Co.,, analytical pure).
(10) Trisodium Citrate (China Medicine (Group) Shanghai Chemical Reagent Co.,, analytical pure): use the deionized water recrystallization before using.
(11) Tetramethyl Ethylene Diamine (TEMED) (China Medicine (Group) Shanghai Chemical Reagent Co.,, analytical pure).
(12) Span 80 (Fluka company).

Claims (3)

1. polymkeric substance/gold (Au) nanoparticle complex media that is used for capillary electrophoresis DNA sequencing is combined by accurate interpenetrating polymer networks and golden nanometer particle, it is characterized in that the viscosity-average molecular weight of linear polyacrylamide: 0.9 * 10 6～3.5 * 10 6Da, acrylamide: N,N-DMAA=10: 1～100: 1 (mol ratio), the diameter of Au are 10～65nm, Au content is 20～1200 μ g in every gram polymer/gold nano particle composite medium.
2. a kind of polymer/gold nano particle composite medium that is used for capillary electrophoresis DNA sequencing as claimed in claim 1, wherein the viscosity-average molecular weight of linear polyacrylamide is 3.0 * 10 6Da.
3. the polymer/gold nano particle composite medium that is used for capillary electrophoresis DNA sequencing as claimed in claim 1 is prepared from by following method:
(1) have from applying the synthetic of the accurate interpenetrating polymer networks of function:
The method of at first using reverse emulsion polymerization is in kerosene, Span-80, aqueous systems, obtain linear polyacrylamide with ammonium persulphate/Tetramethyl Ethylene Diamine is acrylamide triggered, the shared mass percent of each component is in whole reaction system: Span-80 is 2～3%, kerosene is 37～43%, water is 31～37%, acrylamide is 20～26%, and ammonium persulphate is 0.002～0.008%, and Tetramethyl Ethylene Diamine is 0.002～0.005%; Concrete reactions steps is: Span-80 is mixed with kerosene, after stirring, logical nitrogen adds the solution that acrylamide and water are formed, logical nitrogen 1 hour, under 20 ℃～40 ℃ temperature, after adding the aqueous solution and Tetramethyl Ethylene Diamine of 10% (w/v) ammonium persulphate, reacted 16～30 hours, with products therefrom with acetone immersion, suction filtration, washing, for several times, obtain the linear polyacrylamide of product continuously;
1% (w/v) polyacrylamide solution under stirring, the speed of 100rpm is fed nitrogen after 0.5～2 hour, add N, the N-DMAA, wherein linear polyacrylamide and N, the mass ratio of N-DMAA is 1: 1～5: 1, logical nitrogen is after 5～30 minutes, the ammonium persulfate aqueous solution that adds 10% (w/v), N wherein, the mass ratio of N-DMAA and ammonium persulphate is 1: 0.01～1: 0.04, and the mass ratio of ammonium persulphate and Tetramethyl Ethylene Diamine is 1: 2～2: 1, and-2～2 ℃ were reacted 16～30 hours down, products therefrom is soaked with acetone, suction filtration, washing, continuously for several times;
(2) preparation of Au nanoparticle:
Trisodium Citrate reduction HAuCl with routine 4The aqueous solution makes Au colloidal sol;
(3) accurate interpenetrating polymer networks/gold nano particle composite medium is synthetic:
With above-mentioned accurate interpenetrating polymer networks and Au colloidal sol according to accurate interpenetrating polymer networks: the mixed of Au colloidal sol=1g: 0.5～25mL, use acetone precipitation, filter, drying, obtain accurate interpenetrating polymer networks/gold nano particle composite medium.
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* Cited by examiner, † Cited by third party
Title
郝凌云等.Au-聚丙烯酰胺纳米复合物紫外辐照法的制备及表征.《中国科学技术大学学报》.2001,第31卷(第1期),117-120. *

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