CN1284795C - Magnetic nano particle nucleic acid separator, and its preparing method and use - Google Patents

Magnetic nano particle nucleic acid separator, and its preparing method and use Download PDF

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CN1284795C
CN1284795C CN 03142274 CN03142274A CN1284795C CN 1284795 C CN1284795 C CN 1284795C CN 03142274 CN03142274 CN 03142274 CN 03142274 A CN03142274 A CN 03142274A CN 1284795 C CN1284795 C CN 1284795C
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magnetic nanoparticles
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layer
nucleic acid
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CN1580067A (en
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沈鹤柏
汪友宝
姜继森
杨仲南
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上海师范大学
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Abstract

本发明涉及一种磁性纳米粒子核酸分离器,它具有如下的三层结构:(1)由核壳型磁性纳米粒子构成的内核层;(2)包覆内核层的3-巯基丙基三甲氧基硅烷中间层;(3)包覆中间层的单链DNA外壳层。 The present invention relates to a magnetic nanoparticles nucleic acid splitter, which has the following layer structure: (1) the core layer is composed of a core-shell type magnetic nanoparticles; 3-mercaptopropyl (2) covering the core layer-trimethoxy silane intermediate layer; single-stranded DNA casing layer (3) covering the intermediate layer. 本发明还涉及制备该磁性纳米粒子核酸分离器的方法。 The present invention further relates to a method for producing the magnetic nanoparticles nucleic separator. 本发明的磁性纳米粒子核酸分离器可简单有效地分离目标DNA或RNA。 Magnetic nanoparticles isolated nucleic acids of the present invention can simply and effectively separate the target DNA or RNA.

Description

磁性纳米粒子核酸分离器、及其制法和应用 Magnetic nanoparticles nucleic separator, its preparation method and application

技术领域 FIELD

本发明涉及一种纳米生物分离器,更具体地说,本发明涉及一种磁性纳米粒子核酸分离器。 The present invention relates to a nano-bio-separation, and more particularly, the present invention relates to a nucleic acid splitter magnetic nanoparticles. 本发明还涉及该分离器的制备方法及用途。 The present invention also relates to preparation and use of the separator.

背景技术 Background technique

众所周知,纳米技术领域是当今最热门的科学技术研究领域,将纳米技术应用于生物科学领域中所形成的新兴科学技术—纳米生物技术,是利用纳米技术研究和解决生命科学领域中的重大问题的科学,它正成为当前重要的前沿科学研究领域之一。 As we all know, the field of nanotechnology is the most popular areas of scientific and technological research, nanotechnology will be applied to emerging science and technology in the field of biological sciences formed - nano-biotechnology, nanotechnology is the study of the use and solve major problems in the field of life sciences Science, it is becoming one of the forefront of current scientific research is important.

现在,磁性纳米粒子在生物技术领域中的应用前景日益受到人们的关注。 Now, the prospect of magnetic nanoparticles in the field of biotechnology in increasing people's attention. 磁性纳米粒子经常被用作生物样品磁场分离的分离介质。 Magnetic nanoparticles are frequently used as the biological sample separation medium magnetic separation. 用磁性纳米粒子的分离方法操作简便、所需设备廉价,同时分离速度快,有利于保持样品的生物活性。 The method of separation using magnetic nanoparticles is simple, inexpensive equipment required, while fast separation speed, helps maintain the biological activity of the sample. 目前,运用越来越广泛。 Currently, the use of more and more widely.

用于生物技术中的磁性纳米粒子一般要求具有如下的特性:(1)粒子具有超顺磁性,易于吸附和洗脱;(2)粒子需要较大的磁化强度,以保证分离效率的灵敏度;(3)粒子表面要易于进行化学修饰,以和不同的生物和药物分子进行连接;(4)用于体内时要求有较好的生物相容性。 Magnetic nanoparticles for biotechnology is generally required to have the following characteristics: (1) particles having a superparamagnetic, easy adsorption and elution; (2) requires a large particle magnetization, the sensitivity to ensure the separation efficiency; ( 3) be susceptible to chemical particle surface modification to and different biological molecules and drug connection; (4) the time required for in vivo have good biocompatibility. 基于此目的设计的纳米粒子分离器通常为三明治结构,内核层一般为磁性纳米材料,中间为包覆层,目前已有的产品大多数采用高分子材料作为包覆层,最外层则修饰与生物组织有特异性作用的功能基团,以满足生物分离的要求。 For this purpose a separator designed nanoparticles generally sandwich structure, the core layer is typically a magnetic nano-materials, the intermediate cladding layer is, currently available products as in most cases, a polymer material cladding layer, the outermost layer is modified with biological tissue has a functional group specific effect to meet the requirements of bioseparations.

但是,现有技术中的分离器多采用乳液聚合得到的高分子微球,其中包覆了纳米尺寸的磁性颗粒。 However, the prior art splitter to use more polymer microspheres obtained by emulsion polymerization, wherein the coated magnetic particles of nanometer size. 然而,高分子微球的结构通常比较疏松,其中包含的磁性粒子在长时间保存过程中容易脱离微球并且会产生团聚现象,从而影响产品的性能;在磁性纳米粒子上用吸附或亲合作用固定活性物质时,容易脱落。 However, polymeric microspheres usually loose structure, wherein the magnetic particles contained in the disengaged easily during long-term storage and the microspheres will produce agglomeration, thus affecting the performance of the product; adsorption or affinity in cooperation with magnetic nanoparticles fixed active substance, easily.

本发明提供了一种磁性纳米粒子核酸分离器,克服了现有技术的不足。 The present invention provides a nucleic acid splitter magnetic nanoparticles, overcomes the disadvantages of the prior art.

发明内容 SUMMARY

本发明的目的是提供一种磁性纳米粒子核酸分离器。 Object of the present invention is to provide a magnetic nanoparticles nucleic separator.

本发明的另一个目的是提供制备该磁性纳米粒子核酸分离器的方法。 Another object of the present invention to provide a method for preparing magnetic nanoparticles of the nucleic acid of the separator.

本发明的再一个目的是将该磁性纳米粒子核酸分离器应用于分离目标DNA或RNA的分离。 A further object of the present invention is the isolated nucleic acid of magnetic nanoparticles is applied to the separation of isolated DNA or RNA target.

在本发明的第一方面,提供了一种磁性纳米粒子核酸分离器,它含有如下的三层结构:(1)由核壳型磁性纳米粒子构成的内核层;(2)包覆内核层的MPTS(3-巯基丙基三甲氧基硅烷)中间层;(3)包覆中间层的单链DNA外壳层。 In a first aspect of the present invention, there is provided a magnetic nanoparticles nucleic acid splitter, comprising the following layer structure: (1) the core layer is composed of a core-shell type magnetic nanoparticles; (2) covering the core layer MPTS (3- mercaptopropyl trimethoxysilane) intermediate layer; single-stranded DNA casing layer (3) covering the intermediate layer.

在另一优选例中,所述的核壳型磁性纳米粒子内核层包括内核和外壳。 In another preferred embodiment, the core-shell type magnetic nanoparticles comprising a core layer core and the shell. 所述内核层的内核成分选自:铁的氧化物、镍、镍-铁合金、或其组合。 The core component of the core layer is selected from: oxides of iron, nickel, a nickel - iron alloy, or combinations thereof. 较佳地,所述内核层的内核成分是铁的氧化物。 Preferably the core component, the core layer of iron oxide. 所述内核层的外壳成分选自二氧化硅、琼脂糖、烯烃聚合物、聚丙烯腈、环氧化合物、或其组合。 Housing component of the core layer is selected from silica, agarose, olefin polymers, polyacrylonitrile, epoxy compound, or a combination thereof. 较佳地,所述内核层的外壳成分是二氧化硅。 Preferably the shell component, the core layer is silica.

其中,所述单链DNA外壳层是通过过硫键与中间层连接的单链DNA。 Wherein the shell layer is a single stranded DNA single stranded DNA sulfur bonds via the intermediate layer is connected through.

在本发明的第二方面,提供了一种制备磁性纳米粒子核酸分离器的方法,它包括以下步骤:(1)在含磁性纳米粒子和内核层外壳形成剂(如正硅酸乙酯)的微乳液体系中,通过油包水型反相微乳液法,形成核壳型磁性纳米粒子,所述的核壳型磁性纳米粒子包括内核和外壳,所述的磁性纳米粒子选自:铁的氧化物、镍、镍-铁合金、或其组合;所述的内核层外壳形成剂形成成分选自下组的外壳:二氧化硅、琼脂糖、烯烃聚合物、聚丙烯腈、环氧化合物、或其组合;(2)使用3-巯基丙基三甲氧基硅烷对步骤(1)的核壳型磁性纳米粒子表面进行修饰,得到修饰了巯基的磁性纳米粒子;(3)使用修饰了过硫键的单链DNA对步骤(2)的修饰了巯基的磁性纳米粒子的表面进行修饰,形成磁性纳米粒子核酸分离器。 In a second aspect of the present invention, there is provided a method of magnetic nanoparticles prepared nucleic acid splitter, comprising the steps of: (1) containing magnetic nanoparticles and core shell layer-forming agent (e.g., tetraethyl orthosilicate) is microemulsion system by water-in-oil microemulsion method, a core-shell type magnetic nanoparticles, said nanoparticles comprising a core-shell type magnetic core and the shell, the magnetic nanoparticles are selected from: iron oxide material, nickel, a nickel - iron alloy, or combinations thereof; said housing core layer forming agent selected from the group shell component: silica, agarose, olefin polymers, polyacrylonitrile, epoxy compound, or combinations thereof; (2) using 3-mercaptopropyl trimethoxysilane step (1) the surface of the core-shell type magnetic nanoparticles is modified to obtain magnetic nanoparticles modified with a mercapto group; (3) using a modified over-sulfur bond single-stranded DNA of step (2) modification of the surface of the magnetic nanoparticles modified mercapto group, the magnetic nanoparticles formed nucleic separator.

其中,所述微乳液体系中TritonX-100、正己醇、环己烷按1∶(1~3)∶(4~6)的比例均匀混合。 Wherein said microemulsion system TritonX-100, n-hexanol, cyclohexane press 1: (1-3): (4-6) a ratio uniformly mixed.

在本发明的第三方面,提供了本发明所述的磁性纳米粒子核酸分离器的用途,它被用于分离DNA或RNA。 In a third aspect of the present invention, there is provided the use of magnetic nanoparticles nucleic separator of the present invention, which is used to separate DNA or RNA.

在本发明的第四方面,提供了一种分离核酸的方法,包括步骤:将该磁性纳米粒子核酸分离器加入到含有目标单链DNA或RNA的溶液中,使其在Tris-HCl和MgCl2的混合溶液中,于一定温度下充分杂交后,在外磁场的引导下将目标单链DNA或RNA分离。 In a fourth aspect of the present invention, there is provided a method of isolating nucleic acids, comprising the steps: addition of the magnetic nanoparticles to the solution containing the isolated nucleic acid is single stranded DNA or RNA target in, and allowed to MgCl2 in Tris-HCl mixed solution was thoroughly hybridization at a certain temperature, under the guidance of an external magnetic field to separate the target single-stranded DNA or RNA.

附图说明 BRIEF DESCRIPTION

图1是修饰了巯基的磁性纳米粒子(FSM)的表面增强拉曼效应图,表明巯基被修饰在磁性纳米粒子的表面,而且被修饰在磁性纳米粒子表面的巯基在银的基底上有很好的拉曼增强效应。 Figure 1 is a modification of the surface of the magnetic nanoparticles mercapto group (FSM) Enhanced Raman view showing a mercapto group is modified surface of the magnetic nanoparticles, but is modified in the mercapto surface of the magnetic nanoparticles have on the silver substrate well Raman enhancement effect.

图2是连接了单链DNA的磁性纳米粒子(FSMD)的表面增强拉曼效应图,表明DNA被连接到磁性纳米粒子的表面,而且有较好的拉曼增强效应。 FIG 2 is a single-stranded DNA surface of magnetic nanoparticles (FSMD) connecting the Raman enhancement view showing the surface of the DNA is coupled to magnetic nanoparticles, and better Raman enhancement effect.

具体实施方式 Detailed ways

本发明者经过广泛而深入的研究,发明了在磁性纳米粒子的表面修饰能识别并结合DNA或RNA的物质,然后用这种磁性纳米粒子去捕获目标DNA或RNA,再在外磁场的作用下进行分离的技术。 The present inventors, after extensive and intensive studies, the invention recognizes the surface modified magnetic nanoparticles and to DNA or RNA binding substance, and then use this to capture the magnetic nanoparticles target DNA or RNA, is carried out and then the external magnetic field acting separation technology.

本发明的制备磁性纳米粒子核酸分离器的方法包括以下步骤:(1)制备磁性纳米粒子用二次蒸馏水分别配制FeSO4·7H2O和FeCl3·6H2O的混合溶液及NaOH溶液。 Method for preparing magnetic nanoparticles isolated nucleic acid according to the invention comprises the steps of: (1) Preparation of magnetic nanoparticles prepared in double distilled water FeSO4 · 7H2O and FeCl3 · 6H2O NaOH solution and the mixed solution, respectively. 在铁盐的混合溶液中Fe2+离子的浓度为0.1~0.2mol/l,Fe3+离子的浓度为0.1~0.3mol/l,NaOH溶液的浓度为2~3mol/l。 In a mixed solution of iron salt of Fe2 + ions is 0.1 ~ 0.2mol / l, Fe3 + ion concentration is 0.1 ~ 0.3mol / l, the concentration of the NaOH solution of 2 ~ 3mol / l. 在剧烈搅拌下将体积为混合盐溶液体积一半的NaOH溶液缓慢地滴加到混合盐溶液中。 Under vigorous stirring to a volume of half the volume of mixed salt solution NaOH solution was slowly added dropwise to the mixed salt solution. 将所得到的固体沉淀在40℃~60℃下陈化12h,用二次蒸馏水将沉淀物清洗数次,过滤后再在40℃~80℃的条件下干燥24h,在玛瑙研钵中研磨后即得产物。 After the obtained solid was precipitated at 40 ℃ ~ 60 ℃ aged 12h, double distilled water and the precipitate was washed several times, then filtered and dried 24h at 40 ℃ ~ 80 ℃, and ground in an agate mortar That product was obtained.

(2)二氧化硅在γ-Fe2O3表面的修饰将TritonX-100、正己醇、环己烷按1∶(1~3)∶(4~6)的比例均匀混合,形成透明稳定的微乳液体系。 (2) silica surface modified γ-Fe2O3 will TritonX-100, n-hexanol, cyclohexane press 1: (1-3): (4-6) a ratio uniformly mixed to form a clear stable microemulsion systems . 将上述微乳液体系置于超声波中处理30-60分钟,再向其中加入0.01~1g的γ-Fe2O3(磁性纳米粒子),用超声波处理3分钟后取出上层液倒入三颈烧瓶中,搅拌30~60分钟使之均匀。 The above-described microemulsion was placed in an ultrasonic treatment for 30-60 minutes, to which was added 0.01 ~ 1g of γ-Fe2O3 (magnetic nanoparticles), sonicated for 3 minutes removed the supernatant was poured into a three-necked flask, stirred for 30 to 60 minutes to make it uniform. 取1ml一定浓度的浓氨水用2ml二次蒸馏水稀释,将其缓慢加入到不断搅拌的微乳液中,持续搅拌10~60分钟使氨水均匀分散在微乳液中。 1ml of a certain concentration of concentrated aqueous ammonia was diluted with 2ml double-distilled water, added slowly with constant stirring to the microemulsion, with stirring for 10 to 60 minutes aqueous ammonia is uniformly dispersed in a microemulsion. 1小时后,向微乳液中滴加1~5ml的正硅酸乙酯(内核层外壳形成剂),同时不断地搅拌10小时,并将体系的温度保持在15~40℃之间。 After 1 hour, added dropwise to the microemulsion orthosilicate 1 ~ 5ml (the core shell layer-forming agent), while continuously stirred for 10 hours, the temperature of the system was maintained between 15 ~ 40 ℃. 向体系中加入丙酮使粒子沉淀,或者将体系静置过夜使粒子自然沉淀,使用乙醇清洗粒子。 The system was added acetone to precipitate the particles, or the system was allowed to stand overnight and the particles precipitated naturally, the particles were washed with ethanol. 将清洗后的粒子置于300~700℃的条件下锻烧1~4个小时,收集核壳型磁性纳米粒子。 The calcined particles after washing was placed 1-4 hours at 300 ~ 700 ℃ collected core-shell type magnetic nanoparticles.

(3)用3-巯基丙基三甲氧基硅烷(MPTS)修饰磁性纳米粒子表面取15mg磁性纳米粒子加入2~10ml的乙酸和乙醇的混合液中,用超声波处理20~60分钟;称取0.01~1g MPTS,用超声波处理10~60分钟;将这两种溶液混合均匀,在10~40℃的条件下反应1小时,然后取出粒子并用乙酸和乙醇的混合液清洗3次,接着在100-300℃真空干燥2小时,收集粒子(FSM)。 (3) Take the mixture was added 15mg of magnetic nanoparticles 2 ~ 10ml ethanol and acetic acid with 3-mercaptopropyl trimethoxysilane (of MPTS) surface-modified magnetic nanoparticles, sonicated for 20 to 60 minutes; Weigh 0.01 ~ 1g MPTS, sonicated for 10 to 60 min; the two solutions were uniformly mixed and reacted at 10 to 40 deg.] C for 1 hour, then removed and the particles washed three times with a mixture of acetic acid and ethanol, followed by 100 300 ℃ dried under vacuum for 2 hours to collect particles (FSM).

(4)单链DNA在磁性纳米粒子表面的修饰取一定量的已修饰了过硫键的单链DNA,加入到500μl的NaHCO3和Na2CO3的混合液中,混合均匀后,向该单链DNA的溶液中加入少量FSM,使之分散均匀,并在潮湿的环境中于10~50℃的条件下反应12~36小时(DNA上的过硫键与中间层上的巯基反应,使DNA分子通过过硫键直接连接于中间层)。 (4) a single-stranded DNA in the surface-modified magnetic nanoparticles certain amount of single-stranded DNA has been modified through a sulfur bond, is added to a mixture of NaHCO3 and Na2CO3 in 500μl of mixed uniformly, to the single-stranded DNA for 12 to 36 hours conditions was added a small amount of the FSM, and dispersed uniformly, and at 10 ~ 50 ℃ in a humid environment at (persulfate bonds sulfhydryl reactive on the intermediate layer on the DNA and the DNA molecules through sulfur bonds are directly connected to the intermediate layer). 分离清洗粒子并在20~80℃的条件下真空干燥10小时,然后用二次蒸馏水分散粒子(FSMD),得到磁性纳米粒子核酸分离器。 Separating the particles were washed and dried 10 hours in vacuo at 20 ~ 80 ℃, and then double distilled water dispersed particles (FSMD), to obtain magnetic nanoparticles nucleic separator.

适用于本发明的单链DNA的长度没有特别的限制。 Useful in the present invention, the length of single-stranded DNA is not particularly limited. 通常平均长度约为5~200bp,较佳地约为10~50bp。 The average length of usually about 5 ~ 200bp, more preferably about 10 ~ 50bp.

核壳型磁性纳米粒子内核层的内核成分除了铁的氧化物以外,还有镍(Ni)或者镍(Ni)和铁(Fe)等的合金。 Magnetic core component of the core-shell nanoparticle core layer in addition to iron oxides, and nickel (Ni) or nickel (Ni) and iron (Fe) alloy and the like.

核壳型磁性纳米粒子内核层的外壳成分除了二氧化硅等无机包裹层以外,还有琼脂糖、烯烃聚合物、聚丙烯腈、环氧化合物等有机包裹层。 Housing component core-shell type magnetic nanoparticles core layer in addition to an inorganic silica wrap layer, and the organic layer was wrapped agarose, olefin polymers, polyacrylonitrile, epoxy compounds. 对于选定的外壳成分,可根据现有技术选用合适的内核层外壳形成剂。 For the selected housing components, the choice of suitable core layer-forming agent according to the prior art housing. 例如当外壳成分为二氧化硅时,可选用正硅酸乙酯或其它合适的内核层外壳形成剂。 For example, when the housing component is silicon dioxide, the choice of TEOS or other suitable core shell layer-forming agent.

本发明的主要优点在于: The main advantage of the present invention is that:

(1)使用的磁性纳米粒子是单分散性的,不产生团聚现象,磁性纳米粒子的形状和直径易于控制;(2)能识别目标DNA或RNA的单链DNA通过化学键与磁性纳米粒子连接,牢固性好,不易脱落;(3)与传统的电泳法相比,本发明方法简便而高效。 (1) The magnetic nanoparticles used are monodisperse, no agglomeration of the magnetic nanoparticles shape and diameter easy to control; (2) to identify target DNA or RNA single strand DNA via a chemical bond with magnetic nanoparticles, solid good, easy to fall off; (3) compared with conventional electrophoresis, the method of the present invention is easy and efficient.

下面结合具体的实施例,对本发明作进一步的阐述。 The following embodiments with reference to specific embodiments, the present invention will be further described. 应该明白,本发明不限于这些具体的实施例。 It should be understood that the invention is not limited to these specific embodiments.

实施例1磁性纳米粒子的内核层的制备方法采用改进的化学共沉淀制备磁性粒子的内核层,具体方法如下:用二次蒸馏水分别配制FeSO4·7H2O和FeCl3·6H2O的混合溶液及NaOH溶液。 The method of preparing the core layer of Example 1 of the magnetic nanoparticles prepared using core layer magnetic particles modified chemical co-precipitation, as follows: distilled water with a secondary FeSO4 · 7H2O and FeCl3 · 6H2O NaOH solution and the mixed solution, respectively. 在铁盐的混合溶液中Fe2+离子的浓度为0.1~0.2mol/l,Fe3+离子的浓度为0.1~0.3mol/l,NaOH溶液的浓度为2~3mol/l。 In a mixed solution of iron salt of Fe2 + ions is 0.1 ~ 0.2mol / l, Fe3 + ion concentration is 0.1 ~ 0.3mol / l, the concentration of the NaOH solution of 2 ~ 3mol / l. 在剧烈搅拌下将体积为混合盐溶液体积一半的NaOH溶液缓慢地滴加到混合盐溶液中。 Under vigorous stirring to a volume of half the volume of mixed salt solution NaOH solution was slowly added dropwise to the mixed salt solution. 将所得到的固体沉淀在40℃~60℃陈化12h,用二次蒸馏水将沉淀物清洗数次,过滤后再在40℃~80℃的条件下干燥24h,在玛瑙研钵中研磨后即得产物。 The obtained solid was precipitated at 40 ℃ ~ 60 ℃ aged 12h, double distilled water and the precipitate was washed several times, then filtered and dried 24h at 40 ℃ ~ 80 ℃, and ground in an agate mortar after to give the product.

实施例2核壳型核酸磁性纳米粒子的制备方法将TritonX-100、正己醇、环己烷按1∶2∶5的比例均匀混合,形成透明稳定的微乳液体系。 Preparation Example 2 of magnetic core-shell nanoparticles to the nucleic acid TritonX-100, n-hexanol, cyclohexane proportion 1:2:5 uniformly mixed to form a clear stable microemulsion systems. 将上述微乳液体系置于超声波中处理30~60分钟,再向其中加入0.5g的γ-Fe2O3,用超声波处理6分钟后取出上层液倒入三颈烧瓶中,搅拌30分钟使之均匀。 The above-described microemulsion was placed in an ultrasonic treatment for 30 to 60 minutes, to which was added 0.5g of γ-Fe2O3, sonicated for 6 minutes removed the supernatant was poured into a three-necked flask, stirred and homogenized for 30 minutes. 取1ml一定浓度的浓氨水用2ml二次蒸馏水稀释,30分钟后将其缓慢加入到不断搅拌的微乳液中,持续搅拌30分钟使氨水均匀分散在微乳液中。 1ml of a certain concentration of concentrated aqueous ammonia was diluted with 2ml double-distilled water 30 minutes after which was slowly added to the microemulsion with constant stirring, for 30 minutes to stirred aqueous ammonia is uniformly dispersed in a microemulsion. 1小时后,向微乳液中滴加1~3ml的正硅酸乙酯,同时不断地搅拌10小时,并将体系的温度保持在15~30℃之间。 After 1 hour, added dropwise to the microemulsion orthosilicate is 1 ~ 3ml, while continuously stirred for 10 hours, the temperature of the system was maintained between 15 ~ 30 ℃. 向体系中加入丙酮使粒子沉淀,或者将体系静置过夜使粒子自然沉淀,使用乙醇清洗粒子。 The system was added acetone to precipitate the particles, or the system was allowed to stand overnight and the particles precipitated naturally, the particles were washed with ethanol. 将清洗后的粒子置于400~700℃的条件下,锻烧1~4个小时,收集粒子。 The particles after washing was placed under conditions of 400 ~ 700 ℃, calcined 1 to 4 hours, the particles were collected.

实施例3磁性纳米粒子核酸分离器的制备取15mg实施例2中制得的磁性纳米粒子加入2ml的乙酸和乙醇的混合液中,用超声波处理20~60分钟;称取0.01g MPTS,用超声波处理10~60分钟;将这两种溶液混合均匀,在10~30℃的条件下反应1小时,然后取出粒子并用乙酸和乙醇的混合液清洗3次,接着在100~300℃真空干燥2小时,收集粒子(FSM)。 Example 3 Preparation of magnetic nanoparticles taken 15mg nucleic separator magnetic nanoparticles prepared in Example 2 was added a mixture of ethanol and 2ml of acetic acid, treated with ultrasound 20 to 60 minutes; Weigh 0.01g MPTS, ultrasonically for 10 to 60 minutes; the two solutions were mixed, reacted for 1 hour at 10 ~ 30 ℃ then removed and the particles washed three times with a mixture of acetic acid and ethanol, followed by drying in vacuo at 100 ~ 300 ℃ 2 hours to collect particles (FSM). 从图1中可以看出,巯基被修饰到磁性纳米粒子的表面,而且有很好的拉曼增强效应。 As can be seen from Figure 1, a thiol group is modified to the surface of magnetic nanoparticles, and have a good Raman enhancement effect.

取一定量的已修饰了过硫键的单链DNA(平均长度7bp),加入到500μl的NaHCO3和Na2CO3的混合液中,混合均匀后,向该单链DNA的溶液中加入少量FSM,使之分散均匀,并在潮湿的环境中于20~40℃的条件下反应24小时。 Certain amount of that has been modified through a sulfur bond single-stranded DNA (average length 7bp), was added to a 500μl mixture of Na2CO3 and NaHCO3, the uniformly mixed to single-stranded DNA was added a small amount of the FSM, so uniformly dispersed, and under 20 ~ 40 ℃ in 24 hours in a humid environment. 分离清洗粒子并在20~80℃的条件下真空干燥10小时,然后用二次蒸馏水分散粒子(FSMD),得到磁性纳米粒子核酸分离器。 Separating the particles were washed and dried 10 hours in vacuo at 20 ~ 80 ℃, and then double distilled water dispersed particles (FSMD), to obtain magnetic nanoparticles nucleic separator. 从图2中可以看出,DNA探针被连接到了磁性纳米粒子表面,而且有明显的拉曼增强效应。 As can be seen from Figure 2, DNA probes are connected to the surface of the magnetic nanoparticles, and a clear enhancement of the Raman effect.

实施例4磁性纳米粒子核酸分离器的制备取15mg实施例2制得的磁性纳米粒子加入6ml的乙酸和乙醇的混合液中,用超声波处理20-60分钟;称取0.5g MPTS,用超声波处理10~60分钟;将这两种溶液混合均匀,在10~30℃的条件下反应1小时,然后取出粒子并用乙酸和乙醇的混合液清洗3次,接着在100~300℃真空干燥2小时,收集粒子(FSM)。 Example 4 Preparation of magnetic nanoparticles taken 15mg nucleic separator magnetic nanoparticles prepared in Example 2 was added a mixture of acetic acid and embodiments 6ml of ethanol, sonicated for 20-60 minutes; Weigh 0.5g MPTS, sonicated 10 to 60 min; the two solutions were mixed, reacted at 10 ~ 30 ℃ for 1 hour, then removed and the particles washed three times with a mixture of acetic acid and ethanol, followed by drying in vacuo at 100 ~ 300 ℃ 2 hours. collect particles (FSM).

取一定量的已修饰了过硫键的单链DNA(平均长度50bp),加入到500μl的NaHCO3和Na2CO3的混合液中,混合均匀后,向该单链DNA的溶液中加入少量FSM,使之分散均匀,并在潮湿的环境中于20~40℃的条件下反应24小时。 Certain amount of that has been modified through a sulfur bond single-stranded DNA (average length of 50 bp), was added to a 500μl mixture of Na2CO3 and NaHCO3, the uniformly mixed to single-stranded DNA was added a small amount of the FSM, so uniformly dispersed, and under 20 ~ 40 ℃ in 24 hours in a humid environment. 分离清洗粒子并在20~80℃的条件下真空干燥10小时,然后用二次蒸馏水分散粒子(FSMD),得到磁性纳米粒子核酸分离器。 Separating the particles were washed and dried 10 hours in vacuo at 20 ~ 80 ℃, and then double distilled water dispersed particles (FSMD), to obtain magnetic nanoparticles nucleic separator.

实施例5磁性纳米粒子核酸分离器的制备取15mg实施例2制得的磁性纳米粒子加入10ml的乙酸和乙醇的混合液中,用超声波处理20~60分钟;称取1g MPTS,用超声波处理10~60分钟;将这两种溶液混合均匀,在10~30℃的条件下反应1小时,然后取出粒子并用乙酸和乙醇的混合液清洗3次,接着在100~300℃真空干燥2小时,收集粒子(FSM)。 Example 5 Preparation of magnetic nanoparticles taken 15mg nucleic separator magnetic nanoparticles Example 2 was added 10ml of a mixture of ethanol and acetic acid, treated with ultrasound 20 to 60 minutes; Weigh 1g MPTS, treated with ultrasonic waves 10 to 60 min; the two solutions were mixed, reacted for 1 hour at 10 ~ 30 ℃ then removed and the particles washed three times with a mixture of acetic acid and ethanol, followed by 100 ~ 300 ℃ dried for 2 hours under vacuum, were collected particles (FSM).

取一定量的已修饰了过硫键的单链DNA(平均长度95bp),加入到500μl的NaHCO3和Na2CO3的混合液中,混合均匀后,向该单链DNA的溶液中加入少量FSM,使之分散均匀,并在潮湿的环境中于20~40℃的条件下反应24小时。 Certain amount of that has been modified through a sulfur bond single-stranded DNA (average length 95bp), was added to a 500μl mixture of Na2CO3 and NaHCO3, the uniformly mixed to single-stranded DNA was added a small amount of the FSM, so uniformly dispersed, and under 20 ~ 40 ℃ in 24 hours in a humid environment. 分离清洗粒子并在20~80℃的条件下真空干燥10小时,然后用二次蒸馏水分散粒子(FSMD),得到磁性纳米粒子核酸分离器。 Separating the particles were washed and dried 10 hours in vacuo at 20 ~ 80 ℃, and then double distilled water dispersed particles (FSMD), to obtain magnetic nanoparticles nucleic separator.

实施例6取实施例3~5中制得的磁性纳米粒子核酸分离器,加入100μl总RNA水溶液中,再加入柠檬酸钠缓冲液20~50μl,混匀,在10~50℃下杂交。 Example 6 Examples 3-5 taken prepared magnetic nanoparticles nucleic separator embodiment, the aqueous solution was added 100μl of total RNA, was added sodium citrate buffer 20 ~ 50μl, mixing, hybridization at 10 ~ 50 ℃.

在外在磁场的作用下收集磁性纳米粒子,除去清液。 External magnetic field under the effect of collecting the magnetic nanoparticles, the supernatant was removed. 用柠檬酸钠缓冲液清洗磁性纳米粒子三次。 Washing buffer three times with magnetic nanoparticles sodium citrate. 将磁性纳米粒子悬浮于水中,在外磁场作用下除去磁性纳米粒子,收集水相,并在-80℃的条件下保存备用或用于RT-PCR,cDNA库构建等。 The magnetic nanoparticles suspended in water, removing the external magnetic field of magnetic nanoparticles, aqueous phase was collected and stored for use or used in RT-PCR under the conditions of -80 ℃, cDNA library constructed like.

结果:实施例3-5制得的核壳型核酸磁性纳米粒子是单分散性的,不会产生团聚现象,且其形状和直径易于控制。 Results: Examples 3-5 prepared core-shell type magnetic nanoparticles nucleic acid was monodisperse, the agglomeration does not occur, and it is easy to control the shape and diameter. 此外,能识别目标DNA或RNA的单链DNA通过化学键与该磁性纳米粒子连接,牢固性好,不易脱落,因此分离效果非常好。 Further, to identify target DNA or RNA single strand DNA via a chemical bond to the magnetic nanoparticles, solid good, easy to fall off, and therefore the separation effect is very good.

在不偏离本发明的实质和范围的前提下,本领域技术人员可以对本发明进行各种改动或修改,这些等价的形式同样落在本申请所附权利要求书所限定的范围内。 Without departing from the spirit and scope of the present invention, those skilled in the art that various changes or modifications may be made to the present invention, these equivalent forms also within the present application as defined in the appended claims scope.

Claims (7)

1.一种磁性纳米粒子核酸分离器,其特征在于,它含有如下的三层结构:(1)由核壳型磁性纳米粒子构成的内核层,其中,所述核壳型磁性纳米粒子内核层包括内核和外壳,所述内核层的内核成分选自:铁的氧化物、镍、镍-铁合金、或其组合;所述内核层的外壳成分选自二氧化硅、琼脂糖、烯烃聚合物、聚丙烯腈、环氧化合物、或其组合;(2)包覆内核层的3-巯基丙基三甲氧基硅烷中间层;(3)包覆中间层的单链DNA外壳层。 An isolated nucleic acid of magnetic nanoparticles, characterized in that it comprises the following layer structure: (1) the core layer is composed of a core-shell type magnetic nanoparticles, wherein the core-shell type magnetic nanoparticles core layer comprising core and shell, the core component of the core layer is selected from: oxides of iron, nickel, a nickel - iron alloy, or combinations thereof; the housing component of the core layer is selected from silica, agarose, an olefin polymer, polyacrylonitrile, epoxy compound, or combinations thereof; 3-mercaptopropyl trimethoxysilane the intermediate layer (2) covering the core layer; single-stranded DNA casing layer (3) covering the intermediate layer.
2.根据权利要求1所述的磁性纳米粒子核酸分离器,其特征在于,所述内核层的内核成分是铁的氧化物。 The magnetic nanoparticles isolated nucleic acid of claim 1, wherein the core component of the core layer of iron oxide as claimed in claim.
3.根据权利要求1所述的磁性纳米粒子核酸分离器,其特征在于,所述内核层的外壳成分是二氧化硅。 The magnetic nanoparticles isolated nucleic acid of claim 1, wherein the housing component of the core layer is silica claims.
4.根据权利要求1所述的磁性纳米粒子核酸分离器,其特征在于,所述单链DNA外壳层是通过过硫键与中间层连接的单链DNA。 The magnetic nanoparticles isolated nucleic acid of claim 1, wherein said skin layer is a single stranded DNA single stranded DNA with a sulfur bonds over the intermediate layer connected by the claims.
5.一种制备磁性纳米粒子核酸分离器的方法,其特征在于,它包括以下步骤:(1)在含磁性纳米粒子和内核层外壳形成剂的微乳液体系中,通过油包水型反相微乳液法,形成核壳型磁性纳米粒子,所述的核壳型磁性纳米粒子包括内核和外壳,所述的磁性纳米粒子选自:铁的氧化物、镍、镍-铁合金、或其组合;所述的内核层外壳形成剂形成成分选自下组的外壳:二氧化硅、琼脂糖、烯烃聚合物、聚丙烯腈、环氧化合物、或其组合;(2)使用3-巯基丙基三甲氧基硅烷对步骤(1)的核壳型磁性纳米粒子表面进行修饰,得到修饰了巯基的磁性纳米粒子;(3)使用修饰了过硫键的单链DNA对步骤(2)的修饰了巯基的磁性纳米粒子的表面进行修饰,形成磁性纳米粒子核酸分离器。 A method for preparing magnetic nanoparticles of an isolated nucleic acid, characterized in that it comprises the following steps: (1) in the microemulsion system containing magnetic nanoparticles and core shell layer-forming agent by inverting water-in-oil microemulsion forming core-shell type magnetic nanoparticles, said nanoparticles comprising a core-shell type magnetic core and the shell, the magnetic nanoparticles are selected from: oxides of iron, nickel, a nickel - iron alloy, or combinations thereof; the core layer forming agent component is selected from the casing housing the group consisting of: silica, agarose, olefin polymers, polyacrylonitrile, epoxy compound, or combinations thereof; (2) using 3-mercaptopropyltrimethoxysilane silane of step (1) the surface of the core-shell type magnetic nanoparticles is modified to obtain a modified magnetic nanoparticles and a mercapto group; (3) using a modified over-sulfur bond of single-stranded DNA of step (2) is modified with a mercapto group surface modified magnetic nanoparticles, magnetic nanoparticles are formed nucleic separator.
6.根据权利要求5所述的方法,其特征在于,所述微乳液体系中TritonX-100、正己醇、环己烷按1∶1~3∶4~6的比例均匀混合。 6. The method according to claim 5, wherein said microemulsion system TritonX-100, n-hexanol, cyclohexane 3:4 proportion 1/1 to 1-6 were uniformly mixed.
7.如权利要求1所述的磁性纳米粒子核酸分离器的用途,其特征在于,用于分离DNA或RNA。 7. Use of magnetic nanoparticles according to an isolated nucleic acid as claimed in claim 1, wherein, for isolation of DNA or RNA.
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