CN100348633C - DNA print thermo-sensitive high molecule material and preparation process thereof - Google Patents
DNA print thermo-sensitive high molecule material and preparation process thereof Download PDFInfo
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
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- OAIVIYSBZFEOIU-UHFFFAOYSA-N chloroform;propan-2-one Chemical compound CC(C)=O.ClC(Cl)Cl OAIVIYSBZFEOIU-UHFFFAOYSA-N 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
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- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 abstract description 12
- 239000011159 matrix material Substances 0.000 abstract description 4
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- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 4
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Abstract
本发明以鲑鱼睾DNA印迹热敏性高分子聚N-异丙基丙烯酰胺，合成可对鲑鱼睾DNA分子进行识别的聚合物。首先将模板DNA包埋聚N-异丙基丙烯酰胺中，再利用聚N-异丙基丙烯酰胺的亲疏水性质随温度变化的特征，在交替变化的温度环境下是聚合物在水中膨胀和收缩，促使模板大分子从聚合物母体中洗出，从而得到含DNA印迹空洞的聚合物。该聚合物在液相色谱中显示出了对鲑鱼睾DNA的识别特性。The invention uses salmon testis DNA imprinting heat-sensitive macromolecule poly-N-isopropylacrylamide to synthesize a polymer capable of recognizing salmon testis DNA molecules. First, the template DNA is embedded in poly-N-isopropylacrylamide, and then the characteristics of the hydrophilic and hydrophobic properties of poly-N-isopropylacrylamide vary with temperature. Shrinkage promotes the washing of template macromolecules from the polymer matrix, resulting in polymers containing Southern imprinted cavities. The polymer showed recognition properties for salmon testis DNA in liquid chromatography.
Description
(一)所属技术领域：(1) Technical field:
本发明属于生物大分子印迹技术中DNA印迹聚合物的合成领域，特别是一种DNA印迹热敏高分子材料及其制备方法，该技术是在小分子印迹技术基础上发展起来的，用于制备识别特定DNA分子的新材料，并为解决大分子印迹中模板分子脱出问题提供了一种途径。The invention belongs to the field of synthesis of DNA imprinted polymers in biomacromolecule imprinting technology, in particular to a DNA imprinted thermosensitive polymer material and its preparation method. This technology is developed on the basis of small molecule imprinting technology and is used for the preparation of New materials that recognize specific DNA molecules and provide a way to address the problem of template molecule dropout in macromolecular imprinting.
(二)背景技术：(two) background technology:
DNA印迹聚合物是一种可识别特定DNA大分子的材料。它作为生物大分子印迹技术的发展来源于小分子印迹技术(MIT)(参见Wulff，G.Molecular imprinting in cross-linked materials with the aid of moleculartemplate a way towards artificial antibodies.Angew.Chem.Int.Ed.，1995，34：1821-1832.)。MIT已经广泛应用在手性分离的HPLC固定相、传感器设计、高分子催化剂及人工受体/抗体等领域。其方法是将模板分子溶于或分散于单体溶液中，聚合后的聚合物网络会包埋住模板分子。在强烈的洗涤下，模板分子从聚合物中脱除，聚合物内部便形成了大量与模板分子在形状上互补的空穴，而这些空洞可特异性地结合模板分子。Southern imprinted polymers are materials that recognize specific DNA macromolecules. Its development as biomacromolecule imprinting technology comes from small molecule imprinting technology (MIT) (see Wulff, G. Molecular imprinting in cross-linked materials with the aid of molecular template a way towards artificial antibodies. Angew. Chem. Int. Ed. , 1995, 34: 1821-1832.). MIT has been widely used in the fields of HPLC stationary phase for chiral separation, sensor design, polymer catalyst and artificial receptor/antibody. The method is to dissolve or disperse the template molecules in the monomer solution, and the polymer network after polymerization will embed the template molecules. Under strong washing, the template molecules are removed from the polymer, and a large number of cavities complementary in shape to the template molecules are formed inside the polymer, and these cavities can specifically bind the template molecules.
对于小分子化合物模板，它们易于从聚合物中脱出，形成空洞。聚合物表面印迹蛋白质虽然可使蛋白质从聚合物表面顺利洗脱(参见Shi，H.；Tsai，W.B.；Garrison，M.D.；Ferrari，S.；&Ratner，B.D.Template-imprinted nanostructured surfaces for protein recognition.Nature，1999，398：593-597)，但由于聚合物表面积太小以致不能对大分子进行有效地识别和分离。要获得大的比表面，就必须对聚合物本体内部进行印迹。而大分子印迹的难点就在于如何将生物大分子模板从聚合物内部洗涤出来。因为DNA是生物大分子，其尺寸远大于聚合物的网格，故一旦被包埋就难以从聚合物中脱出。For small molecule compound templates, they are easy to detach from the polymer, forming cavities. Although protein imprinting on the polymer surface can make the protein eluted from the polymer surface smoothly (see Shi, H.; Tsai, W.B.; Garrison, M.D.; Ferrari, S.; & Ratner, B.D. Template-imprinted nanostructured surfaces for protein recognition. Nature, 1999, 398:593-597), but the polymer surface area is too small to effectively recognize and separate macromolecules. To obtain a large specific surface, it is necessary to imprint the interior of the polymer bulk. The difficulty of macromolecular imprinting lies in how to wash out the biomacromolecular template from the inside of the polymer. Because DNA is a biomacromolecule, its size is much larger than the mesh of the polymer, so once it is embedded, it is difficult to escape from the polymer.
(三)发明内容：(3) Contents of the invention:
本发明的目的在于提供一种DNA印迹热敏高分子材料及其制备方法，该方案拟以热敏高分子化合物作为包埋DNA的聚合物，利用交替变化的温度使聚合物的结构经历膨胀和收缩的往复变化，从而促使大分子模板从聚合物内部脱出，这样，就可在一定程度上得到DNA印迹空洞的聚合物，并可在色谱固定相上对DNA分子进行识别。The object of the present invention is to provide a kind of Southern imprint thermosensitive high molecular material and preparation method thereof, this scheme intends to use thermosensitive high molecular compound as the polymer that embeds DNA, utilizes the alternately changing temperature to make the structure of the polymer undergo expansion and The reciprocating change of the shrinkage promotes the release of the macromolecular template from the inside of the polymer, so that the polymer with a Southern imprinting cavity can be obtained to a certain extent, and the DNA molecule can be recognized on the chromatographic stationary phase.
本发明的技术方案：一种DNA印迹热敏高分子材料，其特征在于：该高分子材料具有N-异丙基丙烯酰胺的单体单元结构；该高分子材料是交联型聚合物；该高分子材料结构中含有DNA所印迹的空洞。The technical solution of the present invention: a DNA imprinted thermosensitive polymer material, characterized in that: the polymer material has a monomer unit structure of N-isopropylacrylamide; the polymer material is a cross-linked polymer; The polymer material structure contains cavities imprinted by DNA.
上述所说的DNA印迹热敏高分子材料，随着温度的变化聚合物在水溶液中的体积会发生变化。For the aforementioned Southern imprinted thermosensitive polymer material, the volume of the polymer in the aqueous solution will change as the temperature changes.
上述所说的DNA印迹热敏高分子材料，其聚合物不溶于任何溶剂。In the aforementioned Southern imprint thermosensitive polymer material, the polymer thereof is insoluble in any solvent.
一种DNA印迹热敏高分子材料的制备方法，其特征在于它是由以下步骤构成：A method for preparing a Southern imprinted thermosensitive polymer material is characterized in that it consists of the following steps:
(1)聚合物的制备：在10～20mL 50μg/mL的鲑鱼睾DNA的水溶液中加入5～10g的N-异丙基丙烯酰胺和2～4g的N，N′-亚甲基双丙烯酰胺；溶液在5℃下孵化1～3h，以便在聚合前用DNA分子对单体进行定向；在氮气保护下，以0.35～0.5g的过硫酸钾-亚硫酸氢钠引发自由基聚合反应，在5℃下强烈搅拌，直至体系交联固化；挤压碎胶冻体通过200目的筛网；(1) Preparation of polymer: Add 5-10g of N-isopropylacrylamide and 2-4g of N,N'-methylenebisacrylamide to 10-20mL of 50μg/mL aqueous solution of salmon testis DNA ; The solution was incubated at 5°C for 1 to 3 hours in order to orientate the monomers with DNA molecules before polymerization; under the protection of nitrogen, 0.35 to 0.5 g of potassium persulfate-sodium bisulfite was used to initiate free radical polymerization. Stir vigorously at 5°C until the system is cross-linked and solidified; squeeze the crushed jelly through a 200-mesh sieve;
(2)模板的脱出：以50℃和10℃的蒸馏水交替洗涤聚合物各3～5次，在用索氏提取器萃取1～3h，尽可能将DNA从聚合物中除去；(2) Detachment of the template: alternately wash the polymer with distilled water at 50°C and 10°C for 3 to 5 times, extract with a Soxhlet extractor for 1 to 3 hours, and remove DNA from the polymer as much as possible;
(3)模板脱出量的检测：每间隔20min，用紫外-可见分光光度计测定280nm处的吸收值，直至吸收值不再增长为止；依照标准曲线，计算最终的DNA浓度；以原始浓度减去终态浓度就可推算出聚合物中印迹洞的数量，以推算DNA分子的脱出量；(3) Detection of template detachment amount: measure the absorbance value at 280nm with a UV-visible spectrophotometer every 20 minutes until the absorbance value no longer increases; calculate the final DNA concentration according to the standard curve; subtract the original concentration The final state concentration can be used to calculate the number of imprinted holes in the polymer to calculate the amount of DNA molecules released;
(4)高性能液相色谱：DNA印迹聚合物在氯仿-丙酮(15∶3体积比)中匀浆，利用气动流体泵，在300巴的压强下，以丙酮为溶剂装载到不锈钢柱内；采用甲醇-醋酸(8∶1体积比)洗提的方法从聚合物中吸附和萃取DNA；在室温下洗提，并用分光光度计在280nm处进行跟踪。(4) High-performance liquid chromatography: the Southern imprinted polymer is homogenized in chloroform-acetone (15:3 volume ratio), and loaded into a stainless steel column with acetone as a solvent by using a pneumatic fluid pump at a pressure of 300 bar; Adsorb and extract DNA from the polymer by eluting with methanol-acetic acid (8:1 volume ratio); elute at room temperature, and track at 280 nm with a spectrophotometer.
本发明的工作原理在于：鲑鱼睾动物DNA是一种脱氧核糖核酸大分子，它作为模板溶于单体N-异丙基丙烯酰胺(NIPAAm)中。NIPAAm与N，N′-亚甲基双丙烯酰胺(EBA)共聚交联，形成聚合物网络，同时DNA大分子包埋其中。The working principle of the present invention is that the salmon testes DNA is a macromolecule of deoxyribonucleic acid, which is dissolved in monomer N-isopropylacrylamide (NIPAAm) as a template. NIPAAm is copolymerized and cross-linked with N, N'-methylenebisacrylamide (EBA) to form a polymer network, and DNA macromolecules are embedded in it.
由于DNA分子三维尺度很大，难以从交联聚合物的分子网络中溢出，不能形成DNA所印迹的聚合物纳米洞。这是大分子印迹聚合物技术上的难题之一。选择热敏高分子化合物聚PNIPAAm来包埋大分子DNA，目的是利用这种聚合物的体积随温度的可变性来促使DNA分子的脱出。PNIPAAm的亲疏水性能在30～35℃有一个转变。把包埋DNA的PNIPAAm反复置于10℃和50℃的水介质环境中，在每种温度下放置1h。使聚合物进行多次亲水和疏水的变换。交联的PNIPAAm在水中具有低温下吸水膨胀，在较高温度下收缩的特点。在吸水膨胀时，聚合物分子网络伸展开来，网格撑大，有利于DNA大分子从网格中扩散。在收缩时，聚合物网络收紧折叠，但网络间的缝隙和缺陷反而被撑大，形成聚合物的空穴和孔道，有利于DNA大分子从孔道中扩散。此外，聚合物网络的反复折叠和伸展，也有利于将DNA大分子从聚合物中挤压出去。Due to the large three-dimensional scale of DNA molecules, it is difficult to overflow from the molecular network of cross-linked polymers, and the polymer nanoholes imprinted by DNA cannot be formed. This is one of the technical challenges of macromolecularly imprinted polymers. The thermosensitive polymer compound polyPNIPAAm is selected to embed macromolecular DNA, and the purpose is to use the variability of the volume of this polymer with temperature to promote the release of DNA molecules. The hydrophilic and hydrophobic properties of PNIPAAm have a transition at 30-35°C. The DNA-embedded PNIPAAm was repeatedly placed in an aqueous environment at 10°C and 50°C for 1 hour at each temperature. The polymer undergoes multiple hydrophilic and hydrophobic switches. The cross-linked PNIPAAm has the characteristics of water absorption expansion at low temperature and contraction at higher temperature in water. When water swells, the polymer molecular network stretches out, and the grid expands, which is conducive to the diffusion of DNA macromolecules from the grid. When shrinking, the polymer network is tightened and folded, but the gaps and defects between the networks are expanded instead, forming cavities and pores in the polymer, which is conducive to the diffusion of DNA macromolecules from the pores. In addition, the repeated folding and stretching of the polymer network is also beneficial for squeezing DNA macromolecules out of the polymer.
基于上述原理，一些DNA大分子会沿着孔道和网格脱离聚合物母体，母体所遗留下的痕迹就可作为识别这一生物大分子的空洞。这些空洞与DNA分子的3D结构在形状上互补。所以，这些印迹的空洞能够识别与模板相同的DNA。Based on the above principles, some DNA macromolecules will break away from the polymer matrix along the channels and grids, and the traces left by the matrix can be used as cavities to identify this biomacromolecule. These cavities are complementary in shape to the 3D structure of the DNA molecule. Therefore, the cavities of these blots recognize the same DNA as the template.
被碾碎、过筛和萃取的聚合物用于高效液相色谱的固定相。装载在分析柱上的DNA印迹的和非印迹PNIPAAm分别来分辨DNA。后者在8.6min时DNA从聚合物中被洗提出来，而前者在相同的保留时间下并没有峰出现，这说明RAN倾向于结合那些在DNA印迹的聚合物。The crushed, sieved and extracted polymers are used as stationary phases in high performance liquid chromatography. Southern blotted and non-blotted PNIPAAm were loaded on analytical columns separately to resolve DNA. The latter DNA was eluted from the polymer at 8.6 min, while the former had no peak at the same retention time, which indicated that RAN tended to bind those polymers in the Southern blot.
本发明的优越性在于：利用热敏高分子材料体积随温度转换这一特征，迫使DNA大分子从高分子材料中的脱出，在一定程度上解决了生物大分子难以从包埋的母体中脱出的问题。The advantage of the present invention lies in: the feature that the volume of the thermosensitive polymer material changes with temperature is used to force the DNA macromolecule to escape from the polymer material, which solves the problem that the biological macromolecule is difficult to escape from the embedded matrix to a certain extent. The problem.
(四)具体实施方式：(4) Specific implementation methods:
实施例1：一种DNA印迹热敏高分子材料，其特征在于：该高分子材料具有N-异丙基丙烯酰胺的单体单元结构；该高分子材料是交联型聚合物；该高分子材料结构中含有DNA所印迹的空洞。Embodiment 1: A kind of Southern imprint thermosensitive polymer material, it is characterized in that: the polymer material has the monomer unit structure of N-isopropylacrylamide; The polymer material is a cross-linked polymer; The polymer material The material structure contains cavities where the DNA is imprinted.
上述所说的DNA印迹热敏高分子材料，随着温度的变化聚合物在水溶液中的体积会发生变化。For the aforementioned Southern imprinted thermosensitive polymer material, the volume of the polymer in the aqueous solution will change as the temperature changes.
上述所说的DNA印迹热敏高分子材料，其聚合物不溶于任何溶剂。In the aforementioned Southern imprint thermosensitive polymer material, the polymer thereof is insoluble in any solvent.
一种DNA印迹热敏高分子材料的制备方法，其特征在于它是由以下步骤构成：A method for preparing a Southern imprinted thermosensitive polymer material is characterized in that it consists of the following steps:
1.聚合物的制备：1. Preparation of polymer:
在10mL 50μg/mL的鲑鱼睾DNA的水溶液中加入5g的N-异丙基丙烯酰胺和2g的N，N′-亚甲基双丙烯酰胺；溶液在5℃下孵化1h，以便在聚合前用DNA分子对单体进行定向；在氮气保护下，以0.35g的过硫酸钾-亚硫酸氢钠引发自由基聚合反应，在5℃下强烈搅拌，直至体系交联固化；挤压碎胶冻体通过200目的筛网；Add 5 g of N-isopropylacrylamide and 2 g of N,N′-methylenebisacrylamide to 10 mL of 50 μg/mL aqueous solution of salmon testis DNA; DNA molecules orient the monomers; under nitrogen protection, 0.35g of potassium persulfate-sodium bisulfite is used to initiate free radical polymerization, and vigorously stirred at 5°C until the system is cross-linked and solidified; crushed jelly is extruded Pass through a 200-mesh sieve;
2.模板的脱出：2. Protrusion of the template:
以50℃和10℃的蒸馏水交替洗涤聚合物各3次，每次1h，尽可能将DNA从聚合物中除去；Wash the polymer alternately with distilled water at 50°C and 10°C for 3 times, each time for 1 hour, to remove DNA from the polymer as much as possible;
3.模板脱出量的检测：3. Detection of template detachment:
每间隔20min，用紫外-可见分光光度计测定280nm处的吸收值，直至吸收值不再增长为止；依照标准曲线，计算最终的DNA浓度；以原始浓度减去终态浓度就可推算出聚合物中印迹洞的数量，以推算DNA分子的脱出量；At intervals of 20 minutes, measure the absorbance at 280 nm with a UV-Vis spectrophotometer until the absorbance no longer increases; calculate the final DNA concentration according to the standard curve; subtract the final concentration from the original concentration to calculate the polymer The number of imprinted holes in the medium is used to calculate the amount of DNA molecules released;
4.高性能液相色谱：4. High performance liquid chromatography:
DNA印迹聚合物在氯仿-丙酮(15∶3体积比)中匀浆，利用气动流体泵，在300巴的压强下，以丙酮为溶剂装载到不锈钢柱内；采用甲醇-醋酸(8∶1体积比)洗提的方法从聚合物中吸附和萃取DNA；在室温下洗提，并用分光光度计在280nm处进行跟踪。The Southern imprinted polymer was homogenized in chloroform-acetone (15:3 volume ratio), and loaded into a stainless steel column with acetone as solvent under a pressure of 300 bar using a pneumatic fluid pump; methanol-acetic acid (8:1 volume ratio) ratio) elution method to adsorb and extract DNA from the polymer; elute at room temperature, and track at 280nm with a spectrophotometer.
实施例2：一种DNA印迹热敏高分子材料，其特征在于：该高分子材料具有N-异丙基丙烯酰胺的单体单元结构；该高分子材料是交联型聚合物；该高分子材料结构中含有DNA所印迹的空洞。Embodiment 2: A kind of Southern imprint thermosensitive polymer material, it is characterized in that: the polymer material has the monomer unit structure of N-isopropylacrylamide; The polymer material is a cross-linked polymer; The polymer material The material structure contains cavities where the DNA is imprinted.
上述所说的DNA印迹热敏高分子材料，随着温度的变化聚合物在水溶液中的体积会发生变化。For the aforementioned Southern imprinted thermosensitive polymer material, the volume of the polymer in the aqueous solution will change as the temperature changes.
上述所说的DNA印迹热敏高分子材料，其聚合物不溶于任何溶剂。In the aforementioned Southern imprint thermosensitive polymer material, the polymer thereof is insoluble in any solvent.
一种DNA印迹热敏高分子材料的制备方法，其特征在于它是由以下步骤构成：A method for preparing a Southern imprinted thermosensitive polymer material is characterized in that it consists of the following steps:
1.聚合物的制备：1. Preparation of polymer:
在20mL 50μg/mL的鲑鱼睾DNA的水溶液中加入10g的N-异丙基丙烯酰胺和4g的N，N′-亚甲基双丙烯酰胺；溶液在5℃下孵化2h，以便在聚合前用DNA分子对单体进行定向；在氮气保护下，以0.5g的过硫酸钾-亚硫酸氢钠引发自由基聚合反应，在5℃下强烈搅拌，直至体系交联固化；挤压碎胶冻体通过200目的筛网；Add 10g of N-isopropylacrylamide and 4g of N,N'-methylenebisacrylamide to 20mL of 50μg/mL aqueous solution of salmon testis DNA; DNA molecules orient the monomers; under the protection of nitrogen, use 0.5g of potassium persulfate-sodium bisulfite to initiate free radical polymerization, and stir vigorously at 5°C until the system is cross-linked and solidified; extrude the crushed jelly Pass through a 200-mesh sieve;
2.模板的脱出：2. Protrusion of the template:
以50℃和10℃的蒸馏水交替洗涤聚合物各5次，在用索氏提取器萃取3h，尽可能将DNA从聚合物中除去；Wash the polymer alternately with distilled water at 50°C and 10°C for 5 times each, and extract with a Soxhlet extractor for 3 hours to remove DNA from the polymer as much as possible;
3.模板脱出量的检测：3. Detection of template detachment:
每间隔20min，用紫外-可见分光光度计测定280nm处的吸收值，直至吸收值不再增长为止；依照标准曲线，计算最终的DNA浓度；以原始浓度减去终态浓度就可推算出聚合物中印迹洞的数量，以推算DNA分子的脱出量；At intervals of 20 minutes, measure the absorbance at 280 nm with a UV-Vis spectrophotometer until the absorbance no longer increases; calculate the final DNA concentration according to the standard curve; subtract the final concentration from the original concentration to calculate the polymer The number of imprinted holes in the medium is used to calculate the amount of DNA molecules released;
4.高性能液相色谱：4. High performance liquid chromatography:
DNA印迹聚合物在氯仿-丙酮(15∶3体积比)中匀浆，利用气动流体泵，在300巴的压强下，以丙酮为溶剂装载到不锈钢柱内；采用甲醇-醋酸(8∶1体积比)洗提的方法从聚合物中吸附和萃取DNA；在室温下洗提，并用分光光度计在280nm处进行跟踪。The Southern imprinted polymer was homogenized in chloroform-acetone (15:3 volume ratio), and loaded into a stainless steel column with acetone as solvent under a pressure of 300 bar using a pneumatic fluid pump; methanol-acetic acid (8:1 volume ratio) ratio) elution method to adsorb and extract DNA from the polymer; elute at room temperature, and track at 280nm with a spectrophotometer.
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| CN1390859A (en) * | 2002-06-26 | 2003-01-15 | 天津大学 | Magnetic compound microsphere of blot gel for biological macromolecular template and its reverse-phase suspension polymerization process for preparing it |
| US20030130462A1 (en) * | 1999-12-03 | 2003-07-10 | Mathias Ulbricht | Method for producing template-textured materials with high binding specificity and selectivity and utilization of said materials |
| US20040192869A1 (en) * | 2003-03-31 | 2004-09-30 | Kulkarni Mohan Gopalkrishna | Polyvalent imprinted polymer and process of preparation thereof |
| CN1631945A (en) * | 2004-11-25 | 2005-06-29 | 上海交通大学 | Preparation method of molecular surface imprinted polymer |
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| US20030130462A1 (en) * | 1999-12-03 | 2003-07-10 | Mathias Ulbricht | Method for producing template-textured materials with high binding specificity and selectivity and utilization of said materials |
| CN1390859A (en) * | 2002-06-26 | 2003-01-15 | 天津大学 | Magnetic compound microsphere of blot gel for biological macromolecular template and its reverse-phase suspension polymerization process for preparing it |
| US20040192869A1 (en) * | 2003-03-31 | 2004-09-30 | Kulkarni Mohan Gopalkrishna | Polyvalent imprinted polymer and process of preparation thereof |
| CN1631945A (en) * | 2004-11-25 | 2005-06-29 | 上海交通大学 | Preparation method of molecular surface imprinted polymer |
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