核酸适配体在固相萃取技术中的研究进展

核酸适配体是一种经由体外指数级富集系统进化技术筛选得到的随机寡核苷酸片段,该寡核苷酸片段能特异性结合靶物质。核酸适配体与固相萃取技术相结合,可以高选择性地应用于复杂样品中痕量组分的萃取、分离、富集和纯化,由此引起了广泛关注。该文综述了基于核酸适配体的固相萃取研究进展,着重评述了核酸适配体固相萃取柱的制备、固相萃取过程、面临的问题和应用前景。     

蛋白质的核酸适配体筛选的研究进展

核酸适配体是通过指数富集系统配体进化(SELEX)筛选获得的,与靶标具有高亲和力和特异性结合的单链DNA或RNA。蛋白质是生命进程中的关键功能分子。近年来,以蛋白质为靶标的适配体筛选在蛋白质相关的基础及应用研究领域受到广泛关注。核酸适配体应用性能的优劣取决于其亲和力、特异性与稳定性。目前,适配体筛选方法的优化主要是提高筛选效率、提升适配体性能及降低筛选成本。适配体主要筛选步骤包括复合物分离、核酸库优化、次级库的富集、适配体序列分析以及亲和力表征等。迄今为止,以蛋白质-核酸复合物的分离为核心步骤的适配体筛选方法有20余种。本文归纳总结了2005年以来以蛋白质为靶标的适配体筛选技术,讨论了各方法的缺陷与局限。介绍了核酸库的设计优化方法、适配体的序列特征,以及常用的亲和力表征方法。     

与草甘膦特异结合的核酸适配体筛选及应用

通过体外指数富集配体系统进化(SELEX)技术,筛选靶向草甘膦核酸适配体A08。使用酶联寡核苷酸测定法(ELONA)和斑点印迹确认草甘膦核酸适配体A08与草甘膦的特异性,未观察到非特异性。基于ELONA平台,草甘膦检测限为4 ng/μL。圆二色谱(CD)实验表明,草甘膦核酸适配体A08形成茎环和分子内G-四链体,可以稳定存在于结合的磷酸盐缓冲溶液中。此外,亲和力实验显示草甘膦与核酸适配体之间具有强的结合力,解离常数(K_d)为38.38±9.094 nmol/L。基于草甘膦核酸适配体A08的ELONA法测定的准确性在真正的草甘膦样品中得到证实。获得的草甘膦核酸适配体A08为制备检测草甘膦试剂盒奠定了坚实的基础。     

不同金属/适配体双功能复合磁性纳米材料的制备及其对外泌体的富集性能

外泌体作为一种细胞外囊泡,其内容物可以反映亲代细胞的重要信息,而自身也具有独特的结构,能够执行特征的生物学功能。基于外泌体的表面化学和生物学特征,制备了不同类型的金属/适配体(Apt)双功能复合磁性纳米材料,并将其应用于外泌体的富集纯化。将适配体和外泌体表面目标膜蛋白的特异性结合性能与以钛、锆为代表的金属氧化物和外泌体磷脂双层膜的特异性亲和作用结合,可极大地提高分离材料对外泌体的分离选择性和富集容量。分别以Fe₃O₄@Zr-MOFs、Fe₃O₄@Zr-Ti-MOFs和Fe₃O₄@TiO₂等金属有机框架(MOFs)/金属氧化物磁性纳米材料为基底,制备对应的双功能MOFs/金属氧化物-适配体复合磁性纳米材料Fe₃O₄@Zr-MOFs-Apt、Fe₃O₄@Zr-Ti-MOFs-Apt和Fe₃O₄@TiO₂-Apt,并进一步对不同材料的外泌体富集性能加以评价。以超速离心法提取的模型外泌体以及尿液为样品,对修饰相同质量适配体和不同含量金属氧化物的双功能材料的富集性能加以对比。将3种双功能磁性纳米材料应用于尿液外泌体的富集,得到的外泌体裂解后经质谱鉴定,分别得到233、343和832个外泌体蛋白。这一结果也表明双功能磁性纳米材料可以充分结合核酸适配体亲和的高选择性和金属氧化物的高富集容量优势,对于复杂生物样品中外泌体的快速、高效分离纯化具有潜在的应用价值,而针对材料制备和分离纯化方法的设计也为新型外泌体富集材料的设计提供了一条可行的新思路。     

SELEX筛选核酸适配体富集水平理论分析

指数富集的配基系统进化(SELEX)方法是一种新颖的筛选技术,能从人工合成的DNA/RNA文库中筛选得到核酸适配体.根据SELEX原理,与靶标亲和性最强的核酸适配体应该具有最高的富集水平.然而,SELEX实验很少出现这种现象.本文采用数学方法,论证了在满足一定实验条件时,与靶标亲和性最强的核酸适配体并不具有最高的富集水平,并指出了较大的靶标浓度、较低的分离效率会促使这种现象的产生.     

毛细管电泳在核酸适配体研究及核酸适配体筛选中的应用

核酸适配体是指通过指数富集配体系统进化(SELEX)技术从随机寡核苷酸文库中筛选得到的高亲和性与特异性的寡核苷酸序列配体。毛细管电泳是高效、快速、低成本的微量分离分析技术。应用毛细管电泳高效、快速筛选核酸适配体是近几年出现的新方法。本文介绍了核酸适配体筛选过程中的主要分离方法如亲和色谱、醋酸纤维素膜、凝胶电泳和磁性分离等方法,并对近年来毛细管电泳在核酸适配体中的亲和作用研究以及用于核酸适配体筛选(CE-SELEX)的主要方法(ECEEM,NECEEM,Non-SELEX和三者比较)和研究进展进行了综述。     

小分子靶标的核酸适配体筛选的研究进展

核酸适配体(aptamer)是通过指数富集配体系统进化(SELEX)技术筛选得到的核糖核酸(RNA)或单链脱氧核糖核酸(ssDNA)。核酸适配体通过高亲和力特异性地识别小分子、蛋白质、细胞、微生物等多种靶标,在生物、医药、食品和环境检测等领域的应用日渐增多。但目前实际可用的核酸适配体有限,其筛选过程复杂,筛选难度大,制约了其应用。与生物大分子、细胞和微生物等靶标不同,小分子靶标与核酸分子的结合位点少、亲和力弱,且靶标通常需要固定在载体上。此外,小分子靶标结合核酸形成的复合物与核酸自身的大小、质量、电荷性质等方面差异较小,二者的分离难度大。故小分子靶标的核酸适配体筛选过程与大分子和细胞等复合靶标相比有明显差异,筛选难度更大。因此需要根据其自身结构特点和核酸适配体的应用目的选定靶标或核酸库的固定方法,优化靶标核酸复合物的分离方法。本文介绍了不同类型小分子(具有基团差异的单分子、含相同基团分子和手性分子等)靶标的选择及其核酸适配体的筛选方法,并对核酸库的设计、与靶标结合的核酸的分离方法和亲和作用表征方法进行了介绍,列出了自2008年以来报道的40余种小分子靶标的核酸适配体序列和复合物的平衡解离常数(K_d)。     

基于核酸外切酶Ⅲ辅助信号放大的荧光法检测四环素

以核酸适配体作为识别单元、核酸外切酶Ⅲ(EXO Ⅲ)作为信号放大元件以及氧化石墨烯(GO)作为信号开关的荧光传感器来检测四环素(TC)。当体系中没有TC存在时,四环素适配体与其互补链(c DNA)杂交形成双链,EXO Ⅲ不能切割5'端标记有荧光基团的单链信号链(SP),加入GO后,信号链被吸附至GO表面并发生荧光淬灭。当体系中有TC存在时,核酸适配体能够识别并且结合TC,促使c DNA与SP链形成双链,这将诱导EXO Ⅲ从信号链3'端对c DNA-SP双链进行切割,释放出荧光基团,游离的荧光基团不能被GO吸附而淬灭,通过连续的酶促切割,得到更多的游离荧光基团,从而使得荧光信号明显增强。建立的荧光法对TC具有较高选择性,检测限(LOD)为671 pmol/L,方法已用于自来水样品中TC的测定。     

基于核酸适配体和纳米粒子的光学探针

核酸适配体(aptamer)是一类通过指数富集的配体系统进化技术(SELEX)经体外筛选得到的单链DNA或RNA。核酸适配体借自身形成的空间结构与靶标分子特异性结合,具有靶分子广、亲和力高、特异性强、易改造修饰等特点,因而在生命科学、临床诊断、药物发现和环境科学等方面得以广泛应用。近年来,核酸适配体与纳米技术结合,并利用纳米材料在光学、磁学、电学、化学及生物学方面表现出的特殊性质,实现了对靶标分子高灵敏度、高选择性、简便快速的识别与检测。本文评述了基于核酸适配体-纳米粒子特性的光学探针在生物大分子、金属离子和有机小分子检测等领域的应用现状与发展趋势,主要包括比色法、荧光光谱法、表面增强拉曼光谱法等。     

细菌的核酸适配体筛选的研究进展

核酸适配体(aptamer)是通过指数富集配体系统进化技术(SELEX)筛选的能够以高亲和力和高特异性识别靶标分子或细胞的核糖核酸(RNA)和单链脱氧核糖核酸(ssDNA)。作为化学抗体,核酸适配体的制备和合成比抗体的成本更低。核酸适配体的靶标范围极其广泛,包括小分子、生物大分子、细菌和细胞等。针对细菌靶标筛选的适配体,目前主要应用于食品、医药和环境中的细菌检测。细菌的核酸适配体筛选可以通过离心法将菌体-适配体复合物与游离的适配体分离,并通过荧光成像、荧光光谱分析、流式细胞仪分选、DNA捕获元件、酶联适配体分析等方法表征适配体与靶标的相互作用。筛选出的适配体可结合生物、化学检测方法用于细菌检测。本文介绍了细菌适配体的筛选和表征方法以及基于适配体的检测方法的最新进展,分析了不同检测方法的利弊,并列出了2011~2015年筛选的细菌的核酸适配体。     

蚕丝蛋白固相萃取分离富集细胞色素c的研究

在特定实验条件下, 蚕丝蛋白对细胞色素c表现出选择性吸附. 以蚕丝蛋白微填充柱为载体, 在流动系统中建立了细胞色素c的分离富集方法, 以分光光度法在410 nm处检测分离富集过程. 在进样流速低于10 μL/s时, 2 mL样品溶液(pH=5.6的水溶液)中5 μg/mL的细胞色素c可被蚕丝蛋白微柱完全吸附, 而在洗脱流速低于15 μL/s时, 200 μL NaCl溶液(1.0 mol/L)可将吸附的细胞色素c完全洗脱, 分离富集系数为10. 用本方法测定细胞色素c的线性范围为1.0~10.0 μg/mL, 检出限为0.33 μg/mL, 精密度RSD为2.5%(5 μg/mL, n=9). 此外, 还采用本文方法对人全血中的蛋白质进行了分离富集, 并用SDS-聚丙烯酰胺凝胶电泳验证了分离后蛋白的纯度.     

On-line system for peptide mapping by capillary electrophoresis at sub-micromolar concentrations

Peptide mapping has been widely used for the identification of modified proteins involved in certain diseases. Despite the fact that capillary electrophoresis (CE) has been shown to be a powerful tool for the separation and detection of tryptic peptide fragments after protein digestion, this technique lacks sensitivity for mapping proteins isolated in very small quantities from biological samples. Consequently, it has been necessary to preconcentrate the protein before adding the proteolytic enzyme for digestion in solution. These experimental steps are quite long, labor intensive and require a lot of sample handling. In this paper, we describe an on-line system allowing digestion of the protein, followed by preconcentration, separation and detection of the tryptic fragments in 4 h. Up to an 800-fold preconcentration factor was achieved for cytochrome c, despite a loss of separation efficiency induced by the multiple-valve design of the system and dispersion of the 60-nl desorption plug. Moreover, our system showed good migration time reproducibility between peptide maps and could be reused for several samples.     

Proteo-dendrimers designed for complementary recognition of cytochrome c: dendrimer architecture toward nanoscale protein complexation

"Proteo-dendrimers" in which polyanionic hepta(glutamic acids), fluorescent zinc porphyrinate cores, hydrophilic polyether surfaces, and nonpeptide hydrophobic dendrons are combined, were developed as a new series of synthetic receptors for protein recognition. They have polyanionic "patch" structures on their surfaces and undergo complementary electrostatic interactions with a positively charged cytochrome c patch, as observed in biological protein-protein recognition systems. Stability constants of the resulting supramolecular complexes were determined in phosphate buffer (pH 7) by monitoring the fluorescence quenching of the zinc porphyrinates. These proteo-dendrimer receptors exhibited higher affinities with cytochrome c proteins in aqueous solutions than with biological cytochrome b5. Furthermore, they effectively blocked complexation of biological cytochrome b5 with cytochrome c, indicating that the proteo-dendrimers and cytochrome b5 similarly occupy the polycationic patch of cytochrome c.     

Free energies for biological electron transfer from QM/MM calculation: method, application and critical assessment

Computer simulations of biological electron transfer reactions are reviewed with a focus on the calculation of reaction free energy (driving force) and reorganization free energy. Then a mixed quantum mechanical/molecular mechanical (QM/MM) approach is described which is designed for computation of these quantities for pure electron transfer reactions with large donor-acceptor separation distances. The method is applied to intra-protein electron transfer in Ru(bpy)(2)(im)His33 cytochrome c and the results compared to experimental data. Several modeling aspects which are important for successful calculation of free energies with QM/MM are discussed in detail.     

Monolithic porous polymer stationary phases in polyimide chips for the fast high-performance liquid chromatography separation of proteins and peptides

Poly(lauryl methacrylate-co-ethylene dimethacrylate) and poly(styrene-co-divinylbenzene) stationary phases in monolithic format have been prepared by thermally initiated free radical polymerization within polyimide chips featuring channels having a cross-section of 200micromx200microm and a length of 6.8cm. These chips were then used for the separation of a mixture of proteins including ribonuclease A, myoglobin, cytochrome c, and ovalbumin, as well as peptides. The separations were monitored by UV adsorption. Both the monolithic phases based on methacrylate and on styrene chemistries enabled the rapid baseline separation of most of the test mixtures. Best performance was achieved with the styrenic monolith leading to fast baseline separation of all four proteins in less than 2.5min. The in situ monolith preparation process affords microfluidic devices exhibiting good batch-to-batch and injection-to-injection repeatability.     

Selective extraction and recovery of cytochrome c by liquid-liquid extraction using a calix[6]arene carboxylic acid derivative

Recently, we reported that a calix[6]arene carboxylic acid derivative can selectively extract the lysine-rich protein cytochrome c by interacting with amino groups on the protein surface. In the present article, quantitative extraction and recovery of cytochrome c using this calix[6]arene carboxylic acid derivative are described. Both adjustment of the pH under acidic conditions and addition of an alcohol are necessary to strip the extracted protein from an organic solution to an aqueous solution. Separation of cytochrome c and lysozyme using the calix[6]arene was achieved under the optimal conditions. In the forward extraction stage, 93% of the cytochrome c was extracted, while lysozyme remained in the solution. In the subsequent stripping stage, the extracted cytochrome c was quantitatively recovered in an aqueous solution. Finally, separation of these proteins, which have similar molecular weights and isoelectric points, was accomplished.     

细胞色素C在聚乙烯氧化物修饰的金电极上的直接电化学反应

本文报道了细胞色素Ｃ在聚乙烯氧化物修饰的金电极上的直接电化学行为，发现ＰＥＯ是细胞色素Ｃ电化学反应的促进剂，ＰＥＯ修饰膜的形态对细胞色素Ｃ电化学反应的可逆性有较大的影响。     

Molecularly imprinted supermacroporous cryogels for cytochrome c recognition

Molecular imprinting is an attractive biomimetic approach that creates specific recognition sites for the shape and functional group arrangement to template molecules. The purpose of this study is to prepare cytochrome c-imprinted poly(hydroxyethyl methacrylate) (PHEMA)-based supermacroporous cryogel which can be used for the separation of cytochrome c from protein mixtures. N-Methacryloyl-(L)-histidinemethylester (MAH) was used as the metal-coordinating monomer. In the first step, Cu(2+) was complexed with MAH, and the cytochrome c imprinted PHEMA (MIP) cryogel was prepared by free radical cryopolymerization initiated by N,N,N',N'-tetramethylene diamine at -12°C. After polymerization is completed, the template cytochrome c molecules were removed from the MIP cryogel using 0.5 M NaCl solution. The maximum cytochrome c binding amount was 126 mg/g polymer. Selective binding studies were performed in the presence of lysozyme and bovine serum albumin. The relative selectivity coefficients of MIP cryogel for cytochrome c/lysozyme and cytochrome c/bovine serum albumin were 1.7 and 5.2 times greater than those of the non-imprinted PHEMA cryogel, respectively. The selectivity of MIP cryogel for cytochrome c was also confirmed with fast protein liquid chromatography. The MIP cryogel could be used many times with no remarkable decrease in cytochrome c binding capacity.     

Displacement chromatography of biomolecules

Displacement chromatography was used for the preparative-scale separation of peptides, antibiotics, and proteins. The feed components were both purified and concentrated during the separation processes. The components of a peptide mixture were separated on a reverse-phase analytical column using 2-(2-butoxyethoxy) ethanol as the displacer. The use of organic modifiers in the carrier along with an elevated column temperature of 45 degrees C enabled the efficient separation of relatively hydrophobic peptides by displacement chromatography. In addition, the throughput of the process was significantly increased by carrying out the separation at an elevated flow-rate with no adverse effect on product purity. The antibiotic cephalosporin C was isolated from impurities in a fermentation broth using 2-(2-butoxyethoxy)ethanol as the displacer along with a step change in column temperature. The proteins cytochrome c and lysozyme were purified on a weak cation-exchanger column using cationic polymers as the displacers. While polymers of 60 and 20 kilodaltons were both found to be good displacers for these proteins, only the lower molecular weight polymer was readily removed from the column by standard regeneration techniques.     

细胞色素C电化学反应的静电作用模型

首次发现,简单的阴离子Ⅰ~-是细胞色素c电化学反应的良好促进剂。在碘离子修饰的金电极上能观察到细胞色素c的准可逆电化学反应。根据细胞色素c的结构特点和碘离子的吸附特性,提出了一个以静电作用为基础的细胞色素c的电化学反应的模型。