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

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 polymer/gold nanoparticle composite medium for capillary electrophoresis DNA sequencing and its preparation method

technical field

The invention relates to a polymer/gold nanoparticle composite medium for capillary electrophoresis DNA sequencing and a preparation method thereof, belonging to the technical field of DNA sequencing and separation biotechnology.

Background technique

Capillary electrophoresis is a well-established method for DNA analysis. For capillary electrophoresis, since the separation medium determines the migration behavior and separation degree of DNA fragments, it is an important factor for DNA separation. Currently, synthetic polymers are the most widely used separation medium for DNA separation. For an effective For separation media, it should have high screening capacity, low viscosity and self-coating function at the same time.

Synthetic polymers commonly used to separate DNA mainly include hydrophilic homopolymers (Heller C., Electrophoresis, 2001, 22: 629-643). For these homopolymers, most of them need to coat the inner wall of the capillary to prevent Except for electroosmotic flow, only a few polymers such as PEO (polyethylene oxide), PDMA (poly N, N-dimethylacrylamide), and PVP (polyvinylpyrrolidone) have the function of self-coating. In comparison, the separation effect of high-molecular-weight linear polyacrylamide (LPA) among homopolymers is currently the best, but it has no self-coating function, and its viscosity is also high at the concentration of DNA sequencing, which makes automatic column packing difficult. . In order to solve this problem, wang et al. (Wang Y, Liang D, Ying Q,.Chu B, Electrophoresis, 2005, 26:126-136) improved the above-mentioned method, and initiated the polymerization of DMA ( N,N-Dimethacrylamide) to obtain quasi-interpenetrating polymer network (quasi-IPN), adjusting the ratio of AA (acrylamide) to DMA found that the less DMA content under the condition of providing sufficient coating ability, the better the separation efficiency good. However, the molecular weight of LPA in the medium used in this method (~10 ⁷ ) is too high. When dissolved in a buffer solution at a certain concentration, the high molecular weight LPA is easy to degrade and its service life (≤4 months) has been greatly affected. In addition, because the molecular weight is too high, the viscosity is too high, so it is necessary to use a high-pressure system to inject or flush out the capillary, which increases the analysis cost on the one hand, and on the other hand, it is difficult to realize the automatic replacement of the sieving medium. Polymer solutions with higher sieving ability and longer DNA read length usually have higher viscosity. Therefore, it is still an important issue for efficient DNA separation to seek low-viscosity sieving media with high sieving ability.

In recent years, adding various additives (such as polyols, clays, gold nanoparticles, latex particles, etc.) to low-viscosity polymer solutions has proved to be a very effective and simple method. The sieving ability at high concentration (without additives) can be achieved at low concentration, which becomes an effective way to improve separation ability without increasing viscosity. Huang et al. (Huang MF, Huang CC, Chang HT. Electrophoresis, 2003, 24: 2896-2902) proposed to use the PEO solution containing GNPs (gold nanoparticles) as a separation medium to separate dsDNA (double-stranded DNA) fragments, in order to avoid Interaction of capillary wall with DNA, capillary was dynamically coated with 5.0% PVP. Due to the extremely low viscosity (<15cP), it is easy to automatically replace the sieving medium. Studies have shown that PEO containing GNPs has the following advantages when used for dsDNA separation: fast, high separation ability, good reproducibility and easy to fill capillary. But also because of the extremely low viscosity, this medium cannot sort DNA.

Contents of the invention

The invention combines inverse emulsion polymerization and solution polymerization to synthesize a quasi-interpenetrating polymer network composed of a hydrophilic polymer with a separation function and a polymer with a self-coating function, and then introduces gold nanoparticles into the polymer.

The purpose of the present invention is to provide a polymer/gold nanoparticle composite medium for DNA sequencing by capillary electrophoresis. This composite medium can sort DNA bases in an empty capillary at a low viscosity and dissolve in buffer The service life of the composite medium in the solution is extended, and the step of coating the inner wall of the capillary is omitted, which greatly simplifies the column making process; gold nanoparticles can form physical cross-linking points in the quasi-interpenetrating polymer network, which strengthens the separation process The effectiveness of the polymer network formed in the medium makes up for the defect that the molecular weight of the polymer in the separation medium is not too high, so that the separation medium obtained has low viscosity, self-coating function, and can form an efficient polymer network to improve DNA Speed and efficiency of isolation and DNA sequencing.

Another object of the present invention is to provide a method for preparing a polymer/gold nanoparticle composite medium for capillary electrophoresis DNA sequencing.

Realize the technical scheme of the present invention:

The invention provides a polymer/gold nanoparticle composite medium for capillary electrophoresis DNA sequencing, which is composed of a quasi-interpenetrating polymer network and gold nanoparticles, and is characterized in that the viscosity-average molecular weight of linear polyacrylamide: 0.9× 10 ⁶ ~3.5×10 ⁶ Da, acrylamide:N,N-dimethylacrylamide=10:1~100:1 (molar ratio), Au diameter is 10~65nm, per gram of polymer/gold nanoparticle The Au content in the composite medium is 20-1200 μg, the buffer solution concentration of the polymer/gold nanoparticle composite medium used for DNA sequencing is 2.0%-3.0%, and the viscosity-average molecular weight of linear polyacrylamide is preferably 3.0×10 ⁶ Da .

The composite medium is prepared by the following method:

(1) Synthesis of quasi-interpenetrating polymer network with self-coating function:

Firstly, in kerosene, Span-80, and water systems, ammonium persulfate/tetramethylethylenediamine is used to initiate acrylamide to obtain linear polyacrylamide by inverse emulsion polymerization. The proportion of each component in the entire reaction system is The mass percentage is: Span-80 is 2-3%, kerosene is 37-43%, water is 31-37%, acrylamide is 20-26%, ammonium persulfate is 0.002-0.008%, tetramethylethylenediamine It is 0.002～0.005%; the specific reaction steps are: mix Span-80 with kerosene, stir with nitrogen gas, add acrylamide and water solution, nitrogen gas for 1 hour, at 20℃～40℃, add 10% (w/v) After the aqueous solution of ammonium persulfate and tetramethylethylenediamine are reacted for 16 to 30 hours, the resulting product is soaked in acetone, suction filtered, and washed several times in succession to obtain the product linear polyacrylamide;

1% (w/v) polyacrylamide aqueous solution is passed through nitrogen gas under stirring at 100rpm for 0.5 to 2 hours, then N,N-dimethylacrylamide is added, wherein linear polyacrylamide and N,N-dimethylacrylamide The mass ratio of methacrylamide is 1:1 to 5:1. After passing nitrogen for 5 to 30 minutes, add 10% (w/v) ammonium persulfate aqueous solution, wherein N,N-dimethylacrylamide and persulfuric acid The mass ratio of ammonium is 1:0.01～1:0.04, the mass ratio of ammonium persulfate and tetramethylethylenediamine is 1:2～2:1, react at -2～2°C for 16～30 hours, and the resulting product Soak in acetone, suction filter, wash, several times in succession;

(2) Preparation of Au nanoparticles:

Au sol was obtained by reducing HAuCl _aqueous solution with conventional sodium citrate;

(3) Synthesis of quasi-interpenetrating polymer network/gold nanoparticles composite media:

Mix the above quasi-IPP network with Au sol according to the ratio of quasi-IPP network: Au sol = 1g: 0.5-25mL, precipitate with acetone, filter and dry to obtain quasi-IPP network/gold nanoparticles Composite medium.

The more specific preparation method of the composite medium is:

(1) Synthesis of quasi-interpenetrating polymer network (quasi-IPN) with self-coating function

The quasi-interpenetrating polymer network is a polymer network composed of two non-crosslinked polymers interpenetrating each other, while the interpenetrating polymer network refers to an interwoven polymer network formed by two or more crosslinked polymers interpenetrating with each other.

Firstly, in kerosene, Span-80, and water systems, ammonium persulfate (APS)/tetramethylethylenediamine (TEMED) is used to initiate acrylamide (AM) to obtain linear polyacrylamide (LPA) by inverse emulsion polymerization. . The mass percentage of each component in the entire reaction system: 31-37% for water, 37-43% for kerosene, 20-26% for AM, 2-3% for Span-80, and 0.002-0.008% for APS , TEMED is 0.002 to 0.005%. The specific reaction steps are as follows: measure and dissolve a certain amount of Span-80 in an appropriate amount of kerosene, pass nitrogen gas to remove oxygen, and mechanically stir at 500 rpm. Weigh a certain amount of acrylamide and dissolve it in an appropriate amount of deionized water. After it is completely dissolved, a colorless transparent solution is obtained. Use a dropping funnel to drop it into a four-necked bottle. The system first appears in an emulsion state, and then gradually turns milky white. After continuous deoxygenation with nitrogen gas for 1 hour, the temperature was raised, and an appropriate amount of APS aqueous solution and TEMED were respectively added with a micro-syringe, and reacted for 16 to 30 hours. After the reaction is finished, drop the reactant into a large amount of acetone under stirring to obtain a white precipitate, and dry it in vacuum at room temperature, then redissolve the product in water, precipitate it with acetone, filter it with suction, and dry the product in vacuum at room temperature, repeat Three times, the product polyacrylamide was obtained, the intrinsic viscosity [η] of the product was determined by Ubbelohde viscometer (0.1mol/L NaCl aqueous solution, 30°C), and the viscosity-average molecular weight M _v of the product was calculated by the Mark-Houwink formula:

[η]＝9.33×10 ^-3 M _v ^0.75 (mL/g)

Then, a monomer with self-coating function (such as DMA, etc.) is added to the aqueous solution of polyacrylamide, and the polymerization is carried out in a way of oxidation-reduction initiation to obtain a quasi-interpenetrating polymer network with self-coating function. The specific reaction steps are as follows: add polyacrylamide aqueous solution into a four-neck bottle, mechanically stir at 50 rpm, and pass nitrogen gas to remove oxygen in the system. After deoxygenation for 1 hour, add an appropriate amount of N,N-dimethylacrylamide with a syringe, continue nitrogen gas for ten minutes, add an appropriate amount of APS aqueous solution and TEMED with a micro-injector, and react at 0°C for 24 hours. After the reaction is finished, drop the reactant into a large amount of acetone under stirring to obtain a white precipitate, soak it in acetone, filter it with suction, and wash it several times in a row. . The ratio of AM and DMA is determined by proton nuclear magnetic resonance spectrum ( ¹ H-NMR);

(2) Preparation of Au nanoparticles

Glassware required for preparation was rinsed with aqua regia, rinsed with deionized water and dried. Add an appropriate amount of HAuCl ₄ aqueous solution into a round bottom flask equipped with a condenser, and heat the solution to boiling under vigorous stirring. An appropriate amount of sodium citrate aqueous solution was quickly added to the above boiling solution, and the color of the solution quickly changed from light yellow to blue, and then to purple red, indicating that gold nanoparticles (GNPs) were generated. After continuing to boil for 10 minutes, remove the heat source. The sol was then stirred for 15 min and then cooled to room temperature. The size of GNPs can be measured by transmission electron microscopy.

(3) Synthesis of quasi-interpenetrating polymer network/gold nanoparticles composite media

Add the above-mentioned Au sols into appropriate amount of quasi-IPN aqueous solution respectively. After complete mixing and homogeneity, this solution was precipitated with excess acetone, filtered and vacuum dried to obtain a polymer/gold nanoparticle composite medium.

The reaction process is as follows:

Beneficial effects of the present invention: the present invention combines inverse emulsion polymerization and solution polymerization to synthesize the hydrophilic polymer (such as polyacrylamide, etc.) with separation function and the polymer (such as poly N, N-dimethyl A quasi-interpenetrating polymer network composed of acrylamide, etc.), and then introduce gold nanoparticles into this polymer network to form a polymer/gold nanoparticle composite medium. This medium can form a quasi-interpenetrating network with gold nanoparticles as physical crosslinking points at a certain concentration in the buffer solution. Polyacrylamide with separation function, and when the molecular weight of polyacrylamide is very large (~10 ⁷ ), it is easy to degrade in the buffer solution and reduce the separation efficiency. Therefore, due to the molecular weight of the separation polymer (LPA) used in this project (~ 10 ⁶ ) is not very high, so at the concentration of DNA sequencing (2.0%-3.0%), the viscosity is not high (the intrinsic viscosity of the medium [η] ≈ 700mL/g), and the commercial instrument ABI-310 can be used for automatic sequencing; However, in the quasi-interpenetrating network composed of LPA and PDMA in the background technology, when the molecular weight of LPA is close to 10 ⁷ , the viscosity is higher (the intrinsic viscosity of the medium [η ]≈1500mL/g), because it requires a high-pressure system for sample injection, it is difficult to automatically pack the column. In this way, on the one hand, the viscosity of the separation medium can be reduced at a certain concentration, which is convenient for injecting and flushing out of the capillary, and on the other hand, it reduces the possibility of polymer degradation during the preservation of the separation medium solution; The existence of the capillary can prevent the generation of electroosmotic flow without pre-coating the inner wall of the capillary, which saves the step of coating the inner wall of the capillary, greatly simplifies the column making process, reduces the cost of using the capillary, and increases the service life of the capillary; Gold nanoparticles can act as physical crosslinks of the network in the quasi-interpenetrating network, which enhances the effectiveness of the polymer network formed during the separation process and compensates for the efficiency brought about by the less high molecular weight of the main polymer in the separation material Reduced defects, prolong the storage time of the separation medium solution, the storage time of the separation medium in the buffer solution is more than 8 months, and the storage time of high molecular weight (~10 ⁷ ) polymers in the buffer solution is less than 4 months . The service life of general commercial media is 6 months; Compared with the PEO/Au composite system in the background technology that can only separate dsDNA fragments, this separation medium can carry out ssDNA under a certain concentration (2.0%～3.0%) Sort. Through this project, a low-viscosity, self-coating and high-efficiency separation medium can be obtained. Through the study of the separation mechanism, the relationship between the speed and efficiency of DNA separation and sequencing can be fundamentally understood.

Description of drawings

Figure 1: is the schematic diagram of the synthesis of polymer/gold nanoparticles composite media;

Figure 2: It is the sequence diagram of base A of standard sample DNA (Bigdye Terminator V 3.1 sequencing standard) in 1×TTE/7M urea solution of 2.5% (w/v) quasi-IPN/GNPs-1;

Figure 3: 2.5% (w/v) quasi-IPN/GNPs-1 1 × TTE/7M urea solution for the partial sequencing of the four bases of the standard sample DNA (Bigdye Terminator V 3.1 sequencing standard);

Figure 4: It is the sequence diagram of base A of standard sample DNA (Bigdye Terminator V 3.1 sequencing standard) in 1×TTE/7M urea solution of 2.5% (w/v) quasi-IPN/GNPs-2;

Figure 5: It is the sequence diagram of base A of standard sample DNA (Bigdye Terminator V 3.1 sequencing standard) in 1×TTE/7M urea solution of 2.5% (w/v) quasi-IPN/GNPs-3;

Figure 6: It is the sequence diagram of base A of standard sample DNA (Bigdye Terminator V 3.1 sequencing standard) in 1×TTE/7M urea solution of 3.0% (w/v) quasi-IPN/GNPs-4;

Figure 7: It is the sequence diagram of base A of standard sample DNA (Bigdye Terminator V 3.1 sequencingstandard) in 1×TTE/7M urea solution of 2.5% (w/v) quasi-IPN/GNPs-5;

Figure 8: It is the sequence map of base A of standard sample DNA (Bigdye Terminator V 3.1 sequencing standard) in 1×TTE/7M urea solution of 2.0% (w/v) quasi-IPN/GNPs-6.

The following examples further define the present invention, but the present invention is not limited only to the following examples.

Detailed ways

Example 1

(1) Preparation of LPA/PDMA quasi-interpenetrating polymer network

Add Span 80 (2.47g) and refined kerosene (50mL) into a 250mL clean and dry four-neck round bottom flask, and install a mechanical stirrer, reflux condenser, nitrogen inlet/outlet tube and constant pressure funnel on the flask . A solution of acrylamide (AM, 20 g) and H ₂ O (30 g) was added dropwise to the mixture. Under the condition of a mechanical stirring speed of 500 rpm, ultra-high purity nitrogen (UHP, 99.99%) was continuously introduced into the system for 1 h, and then 40 μL of APS (0.1 g/mL) aqueous solution and 4.5 μL of TEMED were added, and reacted at 22 ° C for 24 h. After the polymerization, the emulsion was precipitated in excess acetone, filtered, vacuum-dried, dissolved in water, precipitated with acetone, and vacuum-dried three times to obtain purified linear polyacrylamide (LPA). The viscosity-average molecular weight of LPA measured by Ubbelohde viscometer is 3.0×10 ⁶ Da.

Add 50mL 1% (w/v) LPA aqueous solution into a 100mL clean and dry three-necked flask, pass UHP nitrogen gas, mechanically stir at 100rpm, add 0.2mL DMA at 0°C, add 50μL APS ( 0.1 g/mL) aqueous solution and 8 μL TEMED, after 0.5 h, the stirring speed was adjusted to 50 rpm, and the reaction was continued for 24 h under nitrogen protection. The final product was precipitated with excess acetone, and then dried in vacuo. The result of ¹ H-NMR showed that AM:DMA=30:1.

(2) Preparation of 40nm Au particles

All required glassware was cleaned with aqua regia, rinsed with deionized water and dried. Add 50 mL of HAuCl ₄ (0.01%, wt.) aqueous solution into a round-bottomed flask equipped with a condenser, and heat the solution to boiling under vigorous stirring. Quickly add 0.5mL sodium citrate (1%, wt.) aqueous solution to the above boiling solution, the color of the solution quickly changes from light yellow to blue, and then to purple red, after continuing to boil for 10min, remove the heat source. The sol was then stirred for 15 min and then cooled to room temperature. TEM results show that the average diameter of Au is 40nm.

(3) Preparation and formulation of sieving medium quasi-IPN/GNPs-1

0.25 mL of the above-mentioned Au sol was added to 20 mL of the above-mentioned quasi-IPN aqueous solution of 1% (w/v). After complete mixing, the solution was precipitated with excess acetone, filtered and dried in vacuo.

When performing DNA sequencing experiments, this medium is filled with 1×TTE (50mM Tris (trishydroxymethylaminomethane)/50mM TAPS (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid)/2.0mM EDTA ( EDTA)) buffer solution was dissolved and diluted to 2.5% (concentration unit of polymer solution is w/v).

The conditions for the implementation of capillary electrophoresis are as follows:

Empty capillary effective length/total length: 50/61cm; capillary inner diameter/outer diameter: 75/365um; DNA injection voltage: 2.5KV, injection time: 30s; sequencing voltage: 9.1KV; sequencing temperature: 50°C; DNA sequencing In the process, the buffer solution used in the anode is 1×TTE, and the buffer solution used in the cathode is 1×TTE/7M urea. During the experiment, the empty capillary was first rinsed with deionized water for 60 seconds.

2.5% quasi-IPN/GNPs-1 was used as the sieving medium to obtain the DNA sequencing spectrum, as shown in Fig. 2 and Fig. 3 .

Example 2

(1) Preparation of LPA/PDMA quasi-interpenetrating network

Method is the same as embodiment one.

(2) Preparation of 40nm Au particles

Method is the same as embodiment one.

Preparation and preparation of sieving media quasi-IPN/GNPs-2 with the same particle size and different content of Au

(3) Preparation and formulation of sieving medium quasi-IPN/GNPs-1

Add 2.0 mL of the above-mentioned Au sol to 20 mL of 1% (w/v) above-mentioned quasi-IPN aqueous solution respectively. After complete mixing, the solution was precipitated with excess acetone, filtered and dried in vacuo.

When performing DNA sequencing experiments, the medium was dissolved with 1×TTE buffer solution and diluted to 2.5% (the concentration unit of the polymer solution is w/v).

The implementation conditions of capillary electrophoresis are the same as in Example 1.

2.5% quasi-IPN/GNPs-2 was used as a sieving medium to obtain a DNA sequencing spectrum, as shown in FIG. 4 .

Example 3

(1) Preparation of LPA/PDMA quasi-interpenetrating network

Method is the same as embodiment one.

(2) Preparation of 20nm Au particles

All required glassware was cleaned with aqua regia, rinsed with deionized water and dried. Add 50 mL of 0.01% (wt.) HAuCl ₄ aqueous solution into a round bottom flask equipped with a condenser, and heat the solution to boiling under vigorous stirring. Quickly add 0.85 mL of 1% (wt.) sodium citrate aqueous solution to the above boiling solution, the color of the solution changes from light yellow to blue quickly, and then to purple red. After boiling for 10 minutes, remove the heat source. The sol was then stirred for 15 min and then cooled to room temperature. TEM results show that the average diameter of Au is 20nm.

(3) Preparation and formulation of quasi-IPN/GNPs-3 sieving medium with Au particle size of 20nm

Add 1.0 mL of the above-mentioned Au sol to 20 mL of 1% (w/v) above-mentioned quasi-IPN aqueous solution respectively. After complete mixing, the solution was precipitated with excess acetone, filtered and dried in vacuo.

When performing DNA sequencing experiments, the medium was dissolved with 1×TTE buffer solution and diluted to 2.5% (the concentration unit of the polymer solution is w/v).

The implementation conditions of capillary electrophoresis are the same as in Example 1.

The DNA sequencing spectrum obtained by using 2.5% quasi-IPN/GNPs-3 as the sieving medium is shown in FIG. 5 .

Example 4

(1) Preparation of LPA/PDMA quasi-interpenetrating network

Add Span 80 (2.47g) and refined kerosene (50mL) into a 250mL clean and dry four-neck round bottom flask, and install a mechanical stirrer, reflux condenser, nitrogen inlet/outlet tube and constant pressure funnel on the flask . A solution of acrylamide (AM, 20 g) and H ₂ O (30 g) was added dropwise to the mixture. Under the condition of a mechanical stirring speed of 500 rpm, ultra-high purity nitrogen (UHP, 99.99%) was continuously fed into the system for 1 h, and then 80 μL of APS (0.1 g/mL) aqueous solution and 4.5 μL of TEMED were added, and reacted at 40 ° C for 24 h. After the polymerization, the emulsion was precipitated in excess acetone, filtered, dried in vacuum, then dissolved in water, precipitated with acetone, dried in vacuum, and so on three times to obtain purified LPA. The viscosity-average molecular weight of LPA measured by Ubbelohde viscometer is 0.93×10 ⁶ Da.

Add 50mL of 1% (w/v) LPA aqueous solution to a 100mL clean and dry three-necked flask, pass UHP nitrogen gas, mechanically stir at 100rpm, add 0.30mL DMA at 0°C, and add 100μL APS ( 0.1 g/mL) aqueous solution and 8 μL TEMED, after 0.5 h, the stirring speed was adjusted to 50 rpm, and the reaction was continued for 24 h under nitrogen protection. The final product was precipitated with excess acetone, and then dried in vacuo. The result of ¹ H-NMR showed that AM:DMA=10:1.

(2) Preparation of 40nm Au particles

Method is the same as embodiment one.

(3) Preparation and formulation of quasi-IPN/GNPs-4 sieving medium with Au particle size of 40nm

Add 2.0 mL of the above-mentioned Au sol to 20 mL of 1% (w/v) above-mentioned quasi-IPN aqueous solution respectively. After complete mixing, the solution was precipitated with excess acetone, filtered and dried in vacuo.

When performing DNA sequencing experiments, the medium was dissolved with 1×TTE buffer solution and diluted to 3.0% (the concentration unit of the polymer solution is w/v).

The implementation conditions of capillary electrophoresis are the same as in Example 1.

3.0% quasi-IPN/GNPs-4 was used as a sieving medium to obtain a DNA sequencing spectrum, as shown in FIG. 6 .

Example 5

(1) Preparation of LPA/PDMA quasi-interpenetrating network

Add Span 80 (2.47g) and refined kerosene (50mL) into a 250mL clean and dry four-neck round bottom flask, and install a mechanical stirrer, reflux condenser, nitrogen inlet/outlet tube and constant pressure funnel on the flask . A solution of acrylamide (AM, 20 g) and H ₂ O (30 g) was added dropwise to the mixture. Under the condition of a mechanical stirring speed of 500 rpm, ultra-high purity nitrogen (UHP, 99.99%) was continuously fed into the system for 1 h, and then 80 μL of APS (0.1 g/mL) aqueous solution and 4.5 μL of TEMED were added, and reacted at 40 ° C for 24 h. After the polymerization, the emulsion was precipitated in excess acetone, filtered, dried in vacuum, then dissolved in water, precipitated with acetone, dried in vacuum, and so on three times to obtain purified LPA. The viscosity-average molecular weight of LPA measured by Ubbelohde viscometer is 0.93×10 ⁶ Da.

Add 50mL 1% (w/v) LPA aqueous solution into a 100mL clean and dry three-necked flask, pass UHP nitrogen gas, mechanically stir at 100rpm, add 0.30mL DMA at 0°C, add 15μLAPS (0.1 g/mL) aqueous solution and 8 μL TEMED, after 0.5 h, the stirring speed was adjusted to 50 rpm, and the reaction was continued for 24 h under nitrogen protection. The final product was precipitated with excess acetone, and then dried in vacuo. The result of ¹ H-NMR showed that AM:DMA=100:1.

(2) Preparation of 65nm Au particles

All required glassware was cleaned with aqua regia, rinsed with deionized water and dried. Add 50 mL of 0.01% (wt.) HAuCl ₄ aqueous solution into a round bottom flask equipped with a condenser, and heat the solution to boiling under vigorous stirring. Quickly add 0.35 mL of 1% (wt.) sodium citrate aqueous solution to the above boiling solution, continue boiling for 15 min, and remove the heat source. The sol was then stirred for 15 min and then cooled to room temperature. Transmission electron microscope results show that the average diameter of Au is 65nm.

(3) Preparation and formulation of quasi-IPN/GNPs-5 sieving medium with Au particle size of 65nm

Add 1.0 mL of the above-mentioned Au sol to 20 mL of 1% (w/v) above-mentioned quasi-IPN aqueous solution respectively. After complete mixing, the solution was precipitated with excess acetone, filtered and dried in vacuo.

When performing DNA sequencing experiments, the medium was dissolved with 1×TTE buffer solution and diluted to 2.5% (the concentration unit of the polymer solution is w/v).

The implementation conditions of capillary electrophoresis are the same as in Example 1.

The DNA sequencing spectrum obtained by using 2.5% quasi-IPN/GNPs-5 as the sieving medium is shown in FIG. 7 .

Example 6

(1) Preparation of LPA/PDMA quasi-interpenetrating network

Method is the same as embodiment five.

(2) Preparation of 65nm Au particles

Method is the same as embodiment five.

(3) Preparation and formulation of quasi-IPN/GNPs-6 sieving medium with Au particle size of 65nm

Add 2.0 mL of the above-mentioned Au sol to 20 mL of 1% (w/v) above-mentioned quasi-IPN aqueous solution respectively. After complete mixing, the solution was precipitated with excess acetone, filtered and dried in vacuo.

When performing DNA sequencing experiments, the medium was dissolved with 1×TTE buffer solution and diluted to 2.0% (the concentration unit of the polymer solution is w/v).

The implementation conditions of capillary electrophoresis are the same as in Example 1.

2.5% quasi-IPN/GNPs-6 was used as a sieving medium to obtain a DNA sequencing spectrum, as shown in FIG. 8 .

The reagents used in the above examples were purified according to conventional methods.

(1) Deionized water (Kesheng brand, University of Science and Technology of China): Purify it with SZ-3 automatic triple pure water distiller (Shanghai Huxi Analytical Instrument Factory) before use, and the conductivity of the final water obtained is 1.4× 10 ^-6 S/cm.

(2) Acetone (analytical grade of China Pharmaceutical ₍ Group) Shanghai Chemical Reagent Company): add 0.5g potassium permanganate to 100mL acetone and reflux to remove reducing impurities _. Or CaCO ₃ dry filter, collect 55 ~ 56.5 ℃ fraction.

(3) Chloroform: generally contains 1% ethanol to prevent decomposition into phosgene. Shake it several times with half the volume of water, separate the lower layer of chloroform, dry it with anhydrous calcium chloride for 12 hours, and then distill it. Collect fractions at 61.5-62°C.

(4) Kerosene (commercially available): Wash three times with concentrated sulfuric acid that is one tenth of its volume, and then wash with a saturated solution made of 10% sulfuric acid and potassium permanganate until the purple color of the water layer no longer disappears. Then wash with saturated sodium hydroxide solution, and finally wash with water several times until pH = 7, dry with anhydrous calcium chloride and distill to collect fractions at 180-220°C.

(5) Ammonium persulfate (Aijian Degussa (Shanghai) Initiator Co., Ltd., analytically pure): Prepare a saturated aqueous solution of ammonium persulfate at 30°C, add a little distilled water and filter to remove insoluble matter, and place the solution in the refrigerator After cooling in medium depth, ammonium persulfate crystals were precipitated. Filter, wash with a small amount of deionized water, check whether there is SO ₄ ^2- in the filtrate with BaCl ₂ solution, and crystallize again if necessary. The obtained crystals were vacuum-dried at room temperature, and sealed and stored at 0°C.

(6) Acrylamide (China Pharmaceutical (Group) Shanghai Chemical Reagent Company, analytically pure): 35 g of acrylamide was dissolved in 500 mL of chloroform, heated to 50° C., filtered while hot, cooled slowly at room temperature, and crystals were collected. Dry under vacuum for 2 h at room temperature. Recrystallize once more in the same way, dry in vacuum at room temperature for 24 hours, until the weight difference between the two weighings does not exceed 0.001g, store in a brown bottle and keep it dry in the dark.

(7) N,N-Dimethacrylamide (DMA, Aldrich, ≥99%): distilled under reduced pressure to obtain a colorless transparent liquid, which should be stored at low temperature in the dark.

(8) Tris(hydroxylmethyl)aminomethane, Tris, N-tris(hydroxylmethyl)methyl-3-aminopropanesulfonic acid (N-tris(hydroxylmethyl)methyl-3-aminopropane sulfonic acid , TAPS), ethylenediaminetetraacetic acid (EDTA), urea, analytically pure, all purchased from Aldrich.

(9) HAuCl ₄ ·4H ₂ O (China Pharmaceutical (Group) Shanghai Chemical Reagent Company, analytically pure).

(10) Sodium citrate (China Pharmaceutical (Group) Shanghai Chemical Reagent Company, analytically pure): recrystallized with deionized water before use.

(11) Tetramethylethylenediamine (TEMED) (China Pharmaceutical (Group) Shanghai Chemical Reagent Company, analytically pure).

(12) Span 80 (Fluka Corporation).

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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