CN103304732B - Monodisperse core-shell structure polymer nano particle as well as preparation and application thereof - Google Patents

Monodisperse core-shell structure polymer nano particle as well as preparation and application thereof Download PDF

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CN103304732B
CN103304732B CN201210067274.3A CN201210067274A CN103304732B CN 103304732 B CN103304732 B CN 103304732B CN 201210067274 A CN201210067274 A CN 201210067274A CN 103304732 B CN103304732 B CN 103304732B
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shell
ethanol
boronic acid
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CN103304732A (en
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张丽华
曲焱焱
刘键熙
杨开广
张玉奎
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中国科学院大连化学物理研究所
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Abstract

本发明属高分子材料和分析技术领域,涉及一种单分散核壳结构聚合物纳米粒子及其制备和应用。 The present invention belongs to the technical field of polymer materials and analysis, relates to core-shell polymer nanoparticles and its preparation and use monodisperse. 所述的聚合物纳米粒子是丙烯酸类交联剂与单烯类苯硼酸功能单体的核壳型共聚物,通过“一锅法”,即沉淀聚合结合原位包裹策略制得。 The polymeric nanoparticles are core-shell type acrylic copolymer and a crosslinking agent monoethylenically phenylboronic acid functional monomer, by a "one-pot", i.e. combined situ in the precipitation polymerization prepared policy. 本发明简化了传统核壳结构聚合物粒子的合成步骤,具有快捷易行、成本低廉、表面硼酸功能单体多等特点。 The present invention simplifies conventional synthetic procedures core-shell polymer particles having a quick and easy, low cost, the surface acid functional monomer and more features. 本发明的聚合物纳米粒子可用于分离或富集带有1,2-顺式二醇结构的糖蛋白,在蛋白质组学等领域有较好的实用价值和应用前景。 The polymeric nanoparticles of the present invention may be used to isolate or enrich glycoprotein with a 1,2-cis-diol structure, has good practical value and application prospect in the field of proteomics.

Description

一种单分散核壳结构聚合物纳米粒子及其制备和应用 Monodisperse core-shell polymer nanoparticles and preparation and application

技术领域 FIELD

[0001] 本发明涉及糖蛋白的富集,具体地说是一种单分散核壳结构聚合物纳米粒子及其制备和在糖蛋白富集中应用。 [0001] The present invention relates to enrichment of glycoproteins, in particular a monodisperse core-shell polymer nanoparticles and its preparation and application concentrate rich glycoprotein.

背景技术 Background technique

[0002] 众所周知,蛋白质的组成极其复杂并且不断变化,即使在相同物种的不同细胞或是相同细胞的不同时期,蛋白质的组成都存在显著差异。 [0002] It is well known protein composition is extremely complex and constantly changing, even at different times in different cells of the same species or the same cells, the protein composition are significant differences. 尤其对于翻译后修饰蛋白,往往因其低丰度及复杂性而成为蛋白质组学研宄的热点和难点。 Especially for post-translational modification of proteins, often because of their low abundance and complexity and become a hot and difficult of proteomics research traitor.

[0003] 糖蛋白作为一类重要的翻译后修饰蛋白,在细胞粘附、信号转导、免疫及肿瘤发生等生物学过程中发挥着重要的作用。 [0003] After glycoprotein as an important class translationally modified proteins, it plays an important role in the biological processes of cell adhesion, signal transduction, tumor immunity and the like. 近年来,尽管质谱技术已成为研宄糖蛋白糖基化位点信息及糖型结构归属的有效工具,但由于糖蛋白丰度低、糖肽占总肽段比例小,分析检测时往往被高丰度的非糖肽掩盖,因此在分析之前,需要对糖蛋白/糖肽进行选择性富集。 In recent years, although mass spectrometry has been an effective tool for study based glycoprotein glycosylation site information and attribution saccharide structure, but due to the low abundance of glycoproteins, glycopeptides peptide small proportion of the total, are often high detection analysis abundance non-glycopeptide cover, so before the analysis, the need for glycoproteins / glycopeptides selective enrichment.

[0004]目前最常用的糖蛋白富集方法包括凝集素亲和色谱,酰肼及硼酸功能化材料等。 [0004] The most commonly used method comprises enriching the glycoprotein lectin affinity chromatography, and boric acid hydrazide functional materials. 其中硼酸法是利用糖链上邻二羟基和材料表面硼酸基团间的可逆作用而实现糖蛋白的选择性分离。 Wherein the method is a boric acid selective separation achieved using glycoprotein o- reversible interaction between the material surface and the dihydroxy acid groups on the sugar chain. 此方法步骤简便快捷,避免了糖链结构的破坏,且富集产物与质谱兼容。 This method is simple and quick step, to avoid the destruction of the sugar chain structure, and the enriched product is compatible with mass spectrometry. 有研宄成功的将3-氨基苯硼酸固载于大孔硅球、四氧化三铁颗粒、以及纳米金颗粒表面(Xu Y, WuZ,Zhang L, Lu H,Yang P, Webley PA,Zhao D.Anal.Chem.2009,81,503-508 ;Zhang L,XuY,Yao H,Xie L,Yao J,Lu H,Yang P.Chem.—Eur.J.2009,15,10158-10166 Jang J,LiuY,Qi D,Yao G,Deng C,Zhang X.Proteomics.2009,9,5046-5055),然而所采用的后修饰接枝策略效率较低并且步骤极其繁琐。 There study based on the success of the 3-amino phenyl boronic acid immobilized on a macroporous silica balls, Fe3O4 particles, and nano-gold particle surface (Xu Y, WuZ, Zhang L, Lu H, Yang P, Webley PA, Zhao D.Anal .Chem.2009,81,503-508; Zhang L, XuY, Yao H, Xie L, Yao J, Lu H, Yang P.Chem.-Eur.J.2009,15,10158-10166 Jang J, LiuY, qi D, Yao G, Deng C, Zhang X.Proteomics.2009,9,5046-5055), but the low efficiency of the modified policy adopted by grafting step and extremely cumbersome. 有研宄利用共聚合的方式制备了硼酸功能化整体柱(LRen,Y Liu,M Dong,Z.Liu.J.Chromatogr.A.2009,1216,8421-8425 ;Z Lin,J Pang,HYang,Z Cai, L Zhang, G.Chen, Chem.Commun.2011,47,9675-9677 ;Y Liu, L Ren, Z Liu.Chem.Commun.2011,47,5067-5069),虽硼酸键合效率提高且糖蛋白富集效果较好,但整体柱本身柱容量的限制使得此类材料不利于规模化蛋白样品的预处理。 There study based on the acid functionalized monolithic column (LRen, Y Liu, M Dong, Z.Liu.J.Chromatogr.A.2009,1216,8421-8425 prepared by copolymerizing manner; Z Lin, J Pang, HYang, Z Cai, L Zhang, G.Chen, Chem.Commun.2011,47,9675-9677; Y Liu, L Ren, Z Liu.Chem.Commun.2011,47,5067-5069), although borate bonding efficiency glycoprotein enriched and better, but limits the overall capacity of the column so that the column itself, such materials are not conducive to large-scale protein sample pretreatment.

发明内容 SUMMARY

[0005] 针对以上不足,本发明的目的是提供一种制备简便,具有糖蛋白富集效果且特异性好的单分散核壳结构聚合物纳米粒子。 [0005] For the above insufficiency, the present invention is to provide a process for preparing simple, glycoprotein enriched with good results and specific core-shell polymer monodisperse nanoparticles. 另一个目的是提供一种快捷的“一锅法”核壳结构制备策略。 Another object is to provide an efficient strategy for preparing core-shell structure "one pot."

[0006] 为实现上述目的,本发明采用的技术方案为: [0006] To achieve the above object, the technical solution adopted by the invention is:

[0007] —种单分散核壳结构聚合物纳米粒子,其特征在于:它的粒径为400_700nm,粒度的多分散系数为0.03-0.05 (“多分散系数”为0.03-0.05,其中的多分散系数:指动态光散射(DLS)粒度测试仪测出来的多分散性指数,也可称之为:粒子粒度的多分散性指数) [0007] - monodisperse core-shell polymer nanoparticles, characterized in that: its diameter is 400_700nm, a particle size polydispersity index of 0.03-0.05 ( "polydispersity index" is 0.03-0.05, wherein polydispersity factor: refers to dynamic light scattering (DLS) particle analyzer test out the polydispersity index, also referred to as: particle size polydispersity index)

[0008] 以4-乙烯基苯硼酸、N,f -亚甲基双丙烯酰胺作为单体,在水醇混合体系中通过沉淀聚合的方式首先制得聚合物母核;随后不经过任何离心、洗涤和转移等步骤,在原有聚合条件下继续向体系中添加4-乙烯基苯硼酸功能单体,从而“一锅法”制得表面富含硼酸功能基团的单分散核壳结构聚合物纳米粒子。 [0008] In 4-vinyl phenyl boronic acid, N, f - Methylene bis acrylamide as a monomer, in an aqueous alcohol mixed by a precipitation polymerization system firstly to prepare a polymer nucleus; without any subsequent centrifugation, washing step and metastasis, continue to add 4-vinylphenyl boronic acid functional monomer to the polymerization system in the original condition, so that "pot" boronic produce a surface rich in mono functional group is dispersed core-shell polymer nano particle.

[0009] 所述单分散核壳结构聚合物纳米粒子制备的具体步骤如下, DETAILED Step [0009] The monodisperse particles of core-shell polymer nanoparticles prepared as follows,

[0010] I)形成聚合物核:4-乙烯基苯硼酸、N,N'-亚甲基双丙烯酰胺单体与引发剂分散于水乙醇混合溶剂中,在60-80°C下聚合反应12-24小时制得聚合物核。 [0010] I) formed polymer core: 4-vinyl phenyl boronic acid, N, N'- methylene-bis acrylamide monomer and initiator is dispersed in a mixed solvent of ethanol and water, a polymerization reaction at 60-80 ° C 12-24 hours to prepare a polymer core.

[0011] 2)原位包裹:在所得聚合物核溶液体系中继续加入含4-乙烯基苯硼酸功能单体和引发剂的乙醇溶液,60-80°C下反应4-10小时。 [0011] 2) wrapped in situ: nuclear resultant polymer solution system containing further added 4-vinylbenzyl alcohol acid functional monomer and initiator solutions, the reaction is 4-10 hours at 60-80 ° C.

[0012] 3)离心分离得到核壳结构聚合物纳米粒子,乙醇洗涤数遍,真空干燥至恒重。 [0012] 3) was centrifuged to give core-shell polymer nanoparticles, washed with ethanol several times, and dried in vacuo to constant weight.

[0013] 步骤I)所述的水乙醇混合溶液中乙醇占总溶剂体积的10% -30% ;4_乙烯基苯硼酸与N,N'-亚甲基双丙烯酰胺单体的摩尔比为1: 4-8,单体总摩尔数浓度为0.l-ο.3mol/L ;引发剂为偶氮二异丁腈,其加入量占单体总质量的1_3%。 [0013] Step I) according to a mixed solution of ethanol and water in ethanol 10% -30% of the total solvent volume; 4_ molar ratio of boric acid and divinylbenzene N, N'- methylene-bis-acrylamide monomer 1: 4-8, the total moles of monomer concentration is 0.l-ο.3mol / L; initiator is azobisisobutyronitrile, 1_3% of the total mass of the monomer amount added.

[0014] 步骤2)所述加入的4-乙烯基苯硼酸的摩尔数浓度为0.1-0.3mol/L ;引发剂为偶氮二异丁腈,其质量占后加入4-乙烯基苯硼酸功能单体质量的10-20%;加入终了后,乙醇占水和乙醇所构成的总溶剂体积的20% -40%。 [0014] Step 2) the concentration of added moles of 4-vinyl phenyl boronic acid is 0.1-0.3mol / L; initiator azobisisobutyronitrile, was added after which quality of the 4-vinyl phenyl boronic acid function 10-20% by mass of the monomer; after the addition completion, ethanol 20% -40% of the total volume of solvent composed of water and ethanol.

[0015]制得的单分散核壳结构聚合物纳米粒子用于糖蛋白的选择性富集。 [0015] The resulting single selective enrichment of core-shell polymer dispersed nanoparticles as glycoproteins.

[0016] “一锅法”,即沉淀聚合结合原位包裹策略,作为一种简便快捷的核壳结构聚合物纳米粒子制备方法,往往可以从简单的单体原料出发,无需任何添加剂,并且不经中间体的分离,直接获得核壳结构的聚合物粒子。 [0016] "pot", i.e. combined situ in the precipitation polymerization strategies, as a simple and efficient method for preparing a core-shell structure nano-particles of polymer, often from simple monomer feed, without any additives, and are not the isolated intermediates, obtained directly polymer particles of core-shell structure. 具有反应条件温和,成本低廉以及环境友好等优点,本文正是采用“一锅法”实现了单分散核壳结构聚合物纳米粒子的制备。 Mild reaction conditions, low cost and environmentally friendly, etc., is herein, a "one-pot" method to the preparation of monodisperse core-shell polymer nanoparticles.

[0017] 本发明具有如下优点: [0017] The present invention has the following advantages:

[0018] 1.本发明核壳结构聚合物纳米粒子的粒径分布较窄,实现单分散。 [0018] 1. The particle diameter of the present invention is core-shell polymer nanoparticles distribution is narrow, monodisperse achieved. 颗粒干净光滑,不含有任何添加剂和稳定剂,具有较好的亲水性。 Clear and smooth particles, does not contain any additives and stabilizers, having good hydrophilicity.

[0019] 2.本发明使用两种单体在水醇的混合体系中,利用“一锅法”,得到硼酸功能化核壳结构聚合物纳米粒子;通过活性基团间的偶联,糖蛋白简便并且可逆的结合与粒子表面,实现糖蛋白的选择性富集和MALD1-TOFMS直接分析。 [0019] 2. The present invention uses two monomers mixed system of water-alcohol, using a "one-pot" boronic functionalized core-shell polymer nanoparticles; by coupling between the active groups, glycoproteins easily and reversibly bind to the particle surface, direct analysis of the glycoprotein to achieve selective enrichment and MALD1-TOFMS.

[0020] 3.聚合物核外侧4-乙烯基苯硼酸壳层的形成,使得本发明核壳结构聚合物纳米粒子的表面含有大量硼酸功能基团,并具有更高的糖蛋白吸附容量。 4-vinyl outer core is formed [0020] 3. phenylboronic acid polymer shell, core-shell structure such that the polymer of the present invention the nanoparticle surface containing a large amount of boric acid functional group, and has a higher adsorption capacity glycoprotein.

附图说明 BRIEF DESCRIPTION

[0021] 图1为单分散核壳结构聚合物纳米粒子的合成示意图。 [0021] FIG. 1 is a schematic of the synthesis of monodisperse nanoparticles of core-shell polymer structure.

[0022] 图2a)聚合物母核扫描电镜图;b)单分散核壳结构聚合物纳米粒子扫描电镜图。 [0022] FIG. 2a) polymer nucleus FIG SEM; b) monodisperse core-shell polymer nanoparticles SEM FIG.

[0023]图3单分散核壳结构聚合物纳米粒子的傅里叶红外光谱图。 [0023] FIG. 3 FIG single FTIR dispersed core-shell polymer nanoparticles. 出现了硼酸基团的特征吸收峰(1373和1348cm-1) Boronic acid group appeared characteristic absorption peaks (1373 and 1348cm-1)

[0024] 图4单分散核壳结构聚合物纳米粒子对糖蛋白(辣根过氧化物酶,HRP)与非糖蛋白(牛血清白蛋白,BSA)混合溶液(质量比1:1)的富集效果图。 [0024] FIG. 4 monodispersed core-shell polymer nanoparticles glycoprotein (horseradish peroxidase, HRP) and non-glycoprotein (bovine serum albumin, BSA) mixed solution: Rich (mass ratio 1 1) of FIG diversity effect. a)原液;b)上清;c)富集产物。 a) stock solution; b) the supernatant; c) enriched product.

[0025] 图5单分散核壳结构聚合物纳米粒子对糖蛋白(辣根过氧化物酶,HRP)与非糖蛋白(牛血清白蛋白,BSA)混合溶液(质量比1: 500)的富集效果图。 (Bovine serum albumin, BSA) [0025] FIG. 5 monodisperse core-shell polymer nanoparticles glycoprotein (horseradish peroxidase, HRP) was mixed with non-glycoprotein: Rich (mass ratio of 1 500) FIG diversity effect. a)原液;b)上清;c)富集产物。 a) stock solution; b) the supernatant; c) enriched product.

具体实施方式 Detailed ways

[0026] 下面采用具体实施例对本发明的技术方案做进一步说明。 [0026] The following specific examples further use of the described technical solution of the present invention.

[0027] 实施例1 [0027] Example 1

[0028] 1.单分散核壳结构聚合物纳米粒子的制备 [0028] 1. Single preparing a dispersion of core-shell polymer nanoparticles

[0029] 如图1所示,称取33.8mg 4_乙烯基苯硼酸(VPBA)加入到含有2mL乙醇的50mL圆底烧瓶中,超声5min。 [0029] As shown in FIG 1, divinylbenzene weighed 33.8mg 4_ acid (VPBA) was added to a 50mL round bottom flask containing 2mL of ethanol, the ultrasonic 5min. 待单体溶解后继续向烧瓶中加入8mL水、212.2mg N,f -亚甲基双丙烯酰胺(MBAA)以及5mg偶氮二异丁腈(AIBN),超声直至混合物全部溶解。 After dissolution of the monomers added to the flask to continue 8mL water, 212.2mg N, f - Methylene bis acrylamide (of MBAA) and 5mg of azobisisobutyronitrile (AIBN), ultrasound until the mixture dissolved. 将烧瓶置于油浴中,从室温升温至70°C,并在此温度下维持搅拌、加热18小时。 The flask was placed in an oil bath, heated from room temperature to 70 ° C, stirred and maintained at this temperature, heating for 18 hours. 反应结束后,溶液呈牛奶状,从而制得聚合物母核。 After completion of the reaction, the solution was milky, to produce a polymer nucleus.

[0030] 分别称取33.8mg VPBA和5mg AIBN,超声溶解于2mL乙醇中。 [0030] Weigh and 33.8mg VPBA 5mg AIBN, ultrasound was dissolved in 2mL of ethanol. 将该溶液加入到上述反应结束的聚合体系中,70°C下搅拌、加热6小时,溶液仍呈牛奶状。 This solution was added to the end of the polymerization system in the above reaction, and stirred at 70 ° C, for 6 hours, the solution still on milky. 待反应结束后,停止加热,冷却至室温。 After completion of the reaction, heating was stopped, cooled to room temperature. 将该溶液离心(15000gX20min),弃上清。 The solution was centrifuged (15000gX20min), the supernatant was discarded. 采用乙醇溶液对分离得到的核壳结构聚合物纳米粒子进行洗涤,离心弃上清。 Using an ethanol solution of the core-shell polymer nanoparticles separated was washed, centrifuged supernatant. 重复操作三遍,以去除未反应的单体和反应中形成的低聚物链。 Repeat three times, to remove the oligomer chain reaction and unreacted monomer formed. 真空干燥至恒重,从而制得单分散核壳结构聚合物纳米粒子。 Vacuum dried to a constant weight, to prepare a monodisperse core-shell polymer nanoparticles.

[0031] 2.聚合物纳米粒子的表征 [0031] 2. Characterization of polymeric nanoparticles

[0032] 扫描电镜图如图2所示,沉淀聚合制得的聚合物核的粒径分布较窄,平均粒径为250nm(图2a);原位包裹后形成的核壳结构聚合物粒子的平均粒径为550nm,且表面光滑、单分散、壳层厚度为150nm(图2b)。 [0032] The SEM shown in Figure 2, precipitation polymerization, the polymer core is made narrow particle size distribution, an average particle diameter of 250 nm (FIG. 2A); core-shell polymer particles are formed in situ after the parcel The average particle diameter of 550 nm, and the surface is smooth, monodisperse, shell thickness of 150 nm (FIG. 2b).

[0033] 傅里叶红外光谱表征结果如图3所示,在1373和1348CHT1出现了BO的伸缩振动吸收峰,证明了材料中硼酸基团的存在。 [0033] Fourier transform infrared spectroscopy characterization results shown in Figure 3, the stretching vibration appeared BO absorption peak at 1373 and 1348CHT1, prove the existence of material boronic acid group.

[0034] 实施例2 [0034] Example 2

[0035] 1.蛋白混合溶液配制 [0035] 1. a mixed solution of proteins prepared

[0036] 将糖蛋白(辣根过氧化物酶,HRP)与非糖蛋白(牛血清白蛋白,BSA)按质量比1:1混合,并溶解于50mM碳酸氢钱缓冲溶液中(pH 9.0),从而制得浓度为10ng/μ L的蛋白混合溶液。 [0036] The glycoprotein (horseradish peroxidase, the HRP) and non-glycoprotein (bovine serum albumin, BSA) mass ratio of 1: 1 were mixed and dissolved in 50mM bicarbonate buffer solution money (pH 9.0) to prepare a concentration of 10ng / μ L of protein solution was mixed.

[0037] 2.选择性富集糖蛋白 [0037] 2. Selective enrichment of glycoproteins

[0038] 称取实施例1制备的Img核壳结构聚合物纳米粒子,分散于200 μ L上述蛋白混合溶液中,室温温和震荡2小时,反应结束后离心弃上清。 [0038] Img said core-shell polymer prepared in Example 1 taken embodiment nanoparticles dispersed in 200 μ L of a mixed solution of these proteins, gently shaking at room temperature for 2 hours after the reaction centrifuged supernatant. 50mM碳酸氢铵缓冲溶液(pH 9.0)洗涤材料数遍,离心弃上清。 50mM ammonium bicarbonate buffer solution (pH 9.0) the material was washed several times, centrifuged supernatant. 在分离得到的材料中加入20 μ L含三氟乙酸的50%乙腈溶液(三氟乙酸体积分数I % ),室温温和震荡I小时。 50% acetonitrile was added the material isolated in 20 μ L of trifluoroacetic acid (trifluoroacetate volume fraction I%), I h room temperature with gentle shaking. 离心取上清,直接进行MALD1-TOF MS鉴定。 Centrifuged supernatant, MALD1-TOF MS performed directly identified.

[0039] 3.MALD1-TOF MS 分析 [0039] 3.MALD1-TOF MS Analysis

[0040] 将0.5yL富集产物与0.5yL SA基质(20mg/mL芥子酸溶于含I %三氟乙酸的60%乙腈溶液)依次点于MALDI靶板上,待样品点干燥后进行质谱鉴定。 [0040] The enriched product with 0.5yL SA 0.5yL matrix (20mg / mL sinapinic acid in 60% acetonitrile solution containing I% trifluoroacetic acid) to a point on the MALDI target plate sequentially, to be identified by mass spectrometry after drying the sample point . MALD1-TOF MS实验是在Ultraflex III T0F/T0F(Bruker Daltonics,Bremen,Germany)上进行,检测时米用线性正尚子模式。 MALD1-TOF MS experiments were performed on T0F Ultraflex III / T0F (Bruker Daltonics, Bremen, Germany), when detecting a positive linear meters Naoko mode.

[0041] 如图4所示,经核壳型聚合物纳米粒子富集之后,HRP信号强度较原液显著提高(a曲线);且无BSA非特异吸附,干扰蛋白仅存在于上清之中(b曲线)。 [0041] As shown in FIG. 4, the core-shell polymer nanoparticles after enrichment, signal strength than the HRP stock solution was significantly improved (a curve); BSA non-specific adsorption without interfering proteins present only in the supernatant ( curve b). 表明材料具有较好的糖蛋白富集能力及良好的亲水性(c曲线)。 Indicates that a material having good glycoprotein enriched capacity and good hydrophilicity (c curve).

[0042] 实施例3 [0042] Example 3

[0043] 为了考察聚合物纳米粒子在糖蛋白富集过程中的选择性以及非特异吸附,进一步进行干扰实验。 [0043] In order to examine selectivity of polymeric nanoparticles and non-specific adsorption of the glycoprotein in the enrichment process, further interference experiments. 将HRP和BSA按质量比1: 500混合,并溶解于50mM碳酸氢铵缓冲溶液中(pH 9.0),从而制得浓度为25.05 μ g/ μ L的蛋白混合溶液。 BSA, and HRP-mass ratio of 1: 500 were mixed and dissolved in 50mM ammonium bicarbonate buffer solution (pH 9.0), to prepare a concentration of 25.05 μ g / μ L of protein solution was mixed. 称取Img核壳结构聚合物纳米粒子,以实施例2的条件进行糖蛋白富集及后续MALD1-TOF MS鉴定。 Weigh Img core-shell polymer nanoparticles for glycoprotein enriched and subsequent embodiments MALD1-TOF MS conditions identified in Example 2.

[0044] 如图5所示,原液中HRP由于浓度极低,使得质谱响应信号被BSA抑制而无法检测(a曲线);经核壳型聚合物纳米粒子富集之后,HRP信号强度显著提高(c曲线);且排除了干扰蛋白的影响,使BSA仅在上清之中得到检测(b曲线)。 [0044] 5, the HRP stock solution due to very low concentrations, such that the response signal is suppressed BSA mass can not be detected (a curve); and before the enrichment of core-shell polymer nanoparticles, HRP significantly increased signal intensity ( curve c); and excluding the influence of interfering proteins of BSA only be detected (b curve) in the supernatant. 表明材料对糖蛋白具有极高的选择性。 It indicates that the material is highly selective for the glycoprotein.

Claims (8)

1.一种单分散核壳结构聚合物纳米粒子,其特征在于:它的粒径为400-700nm,粒度的多分散系数为0.03-0.05 ; 以4-乙烯基苯硼酸与N,N' -亚甲基双丙烯酰胺按摩尔比为1:4-8通过沉淀聚合制得的聚合物粒子为核,于核表面原位包裹聚4-乙烯基苯硼酸,形成表面光滑且带有硼酸功能基团的核壳结构的聚合物纳米粒子。 A monodisperse core-shell polymer nanoparticles, characterized in that: its diameter is 400-700 nm, a particle size polydispersity index of 0.03-0.05; to 4-vinyl phenyl boronic acid and N, N '- methylene bis-acrylamide in a molar ratio 1: 4-8 by precipitation polymerization of the polymer particles as a core, the core surface in situ in the poly-4-vinyl phenyl boronic acid, boronic acid is formed having a smooth surface and a functional group polymeric nanoparticle core-shell structure group.
2.按照权利要求1所述单分散核壳结构聚合物纳米粒子,其特征在于:壳的厚度为50-250nm,4-乙烯基苯硼酸在核与壳两相中质量分数之比为15_45%。 2. The according to claim 1 monodisperse core-shell polymer nanoparticles, characterized in that: the shell has a thickness of 50-250nm, 4- divinylbenzene acid than the mass fraction of the two phases of the core and shell 15_45% .
3.—种权利要求1所述单分散核壳结构聚合物纳米粒子的制备方法,其特征在于:以4-乙烯基苯硼酸、N,N' -亚甲基双丙烯酰胺作为单体,在水乙醇混合体系中通过“一锅法”,即沉淀聚合结合原位包裹策略,得到一种具有核壳结构的聚合物纳米粒子。 3.- The single kind of claim 1 preparing core-shell polymer nanoparticle dispersed structure, wherein: the 4-vinyl phenyl boronic acid, N, N '- methylene bis acrylamide as a monomer, in water-ethanol mixture system by "one-pot", i.e. combined situ in the policy precipitation polymerization, to obtain a polymeric nanoparticles having a core-shell structure.
4.按照权利要求3所述的制备方法,其特征在于: 具体步骤如下: 1)形成聚合物核:将4-乙烯基苯硼酸、N,N' -亚甲基双丙烯酰胺单体与引发剂分散于水乙醇混合溶剂中,在60-80°C下聚合反应12-24小时制得聚合物核; 2)原位包裹:在得到的聚合物核溶液体系中继续加入含4-乙烯基苯硼酸功能单体和引发剂的乙醇溶液,60-80°C下反应4-10小时; 3)离心分离得到核壳结构聚合物纳米粒子,用乙醇洗涤,真空干燥至恒重。 4. The method of preparation according to claim 3, characterized in that: the following steps: 1) forming a polymer core: A mixture of 4-vinyl phenyl boronic acid, N, N '- methylene bis acrylamide monomer and initiator dispersed in a mixed solvent of ethanol and water, the polymerization reaction 12-24 hours to prepare a polymer core at 60-80 ° C; 2) wrapped in situ: nuclear system polymer solution obtained was further added a solution of 4-vinyl ethanol solution of phenylboronic acid functional monomer and an initiator, reaction 60-80 ° C 4-10 hours; 3) was centrifuged to give core-shell polymer nanoparticles, washed with ethanol, and dried in vacuo to constant weight.
5.按照权利要求3所述单分散核壳结构聚合物纳米粒子的制备方法,其特征在于:形成聚合物核时期,4-乙烯基苯硼酸与N,N'-亚甲基双丙烯酰胺的摩尔比为1:4-8,单体总摩尔数浓度为0.1-0.3mol/L ;原位包裹时期,于体系中加入4-乙烯基苯硼酸的摩尔数浓度为0.1-0.3mol/Lo 5. according to claim 3 monodisperse core-shell polymer nano particles production method, wherein: during the formation of the polymer core, 4-vinyl phenyl boronic acid and N, N'- methylene bisacrylamide molar ratio of 1: 4-8, on the total moles of monomer concentration 0.1-0.3mol / L; situ in the period, in the system added 4 moles of divinylbenzene acid concentration 0.1-0.3mol / Lo
6.按照权利要求4所述单分散核壳结构聚合物纳米粒子的制备方法,其特征在于:所述引发剂为偶氮二异丁腈,成核时期其加入量占4-乙烯基苯硼酸与N,N' -亚甲基双丙烯酰胺单体总质量的1-3% ;原位包裹时期其加入量占原位包裹时后加入的4-乙烯基苯硼酸功能单体总质量的10-20%。 6. The production method according to claim 4 single core-shell polymer nanoparticle dispersed structure, characterized in that: said initiator is azobisisobutyronitrile, which was added during nucleation 4-vinyl phenyl boronic acid amount and N, N '- methylene bis 1-3% of the total mass of acrylamide monomer; situ in an amount which when added after the addition of 4-vinyl phenyl boronic acid functional monomer situ in the mass of the total period of 10 -20%.
7.按照权利要求4所述单分散核壳结构聚合物纳米粒子的制备方法,其特征在于:成核时期,水醇混合体系中乙醇占总溶剂体积的10% -30% ;原位包裹时期,采用乙醇溶解需加入的4-乙烯基苯硼酸及引发剂,加入终了后,乙醇占水和乙醇所构成的总溶剂体积的20% -40% ο 7. The production method according to claim 4 single core-shell polymer nanoparticle dispersed structure, wherein: the nucleation period, water alcohol mixed system of ethanol 10% -30% of the total solvent volume; situ in Period , dissolved in ethanol to be added using 4-vinyl phenyl boronic acid and an initiator, after the addition of the end, ethanol 20% -40% of the total volume of solvent composed of water and ethanol ο
8.—种权利要求1所述单分散核壳结构聚合物纳米粒子在用于选择性富集糖蛋白上的应用。 8.- The single kind of claim 1 dispersed core-shell polymer nanoparticles applied on a selective enrichment of glycoproteins.
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