Non-SELEX selection of aptamers

Aptamers are typically selected from libraries of random DNA (or RNA) sequences by SELEX, which involves multiple rounds of alternating steps of partitioning and PCR amplification. Here we report, for the first time, non-SELEX selection of aptamers-a process that involves repetitive steps of partitioning with no amplification between them. A highly efficient affinity method, non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), was used for partitioning. We found that three steps of NECEEM-based partitioning in the non-SELEX approach were sufficient to improve the affinity of a DNA library to a target protein by more than 4 orders of magnitude. The resulting affinity was higher than that of the enriched library obtained in three rounds of NECEEM-based SELEX. Remarkably, NECEEM-based non-SELEX selection took only 1 h in contrast to several days or several weeks required for a typical SELEX procedure by conventional partitioning methods. In addition, NECEEM-based non-SELEX allowed us to accurately measure the abundance of aptamers in the library. Not only does this work introduce an extremely fast and economical method for aptamer selection, but it also suggests that aptamers may be much more abundant than they are thought to be. Finally, this work opens the opportunity for selection of drug candidates from libraries of small molecules, which cannot be PCR-amplified and thus are not approachable by SELEX.     

氧化铝富集糖肽的研究

糖基化蛋白对于生命体的生长发育, 免疫调节, 细胞识别粘附等具有重要意义, 而异常的糖基化表达与风湿关节炎、肿瘤、阻塞性肺病等疾病密切相关. 因此糖蛋白结构检测对于研究生命活动至关重要. 由于在复杂样品中糖肽含量相对较少, 加之非糖肽的离子抑制作用, 使得糖肽的质谱检测有一定的挑战性. 因此发展一种有效富集糖肽的方法是必要的. 本实验中我们选用氧化铝对糖肽进行富集研究, 并考察了影响氧化铝保留多肽的机理. 我们利用氧化铝, 从HRP酶解液中共获得16个糖肽, 从IgG酶解液中共获得12个糖肽. 与直接检测样品酶解液和经商品化材料Sepharose富集后再检测相比, 检测到糖肽的个数增多. 实验数据证明氧化铝富集糖肽具有较好的选择性和覆盖率.     

混合模式色谱材料Click TE-GSH高效富集多磷酸化肽

多磷酸蛋白对于生物体适应内外环境具有重要意义,而明确多磷酸蛋白的磷酸位点功能及其信号转导机制尤为关键. 复杂生物样品中多磷酸化肽的低丰度、低电离的特性,以及非磷酸化肽的抑制作用,决定了质谱分析前进行多磷酸化肽富集是非常必要的步骤. 本工作采用基于巯基-烯烃点击化学法合成的混合模式材料Click TE-GSH进行单磷酸化肽和多磷酸化肽的选择性富集. 我们建立了单磷酸化肽、双磷酸化肽和多磷酸化肽的顺序分段富集方法. 该优化方法能抗干扰,应用于脱脂牛奶时富集到11条多磷酸化肽. 与商品化固化金属亲和色谱(IMAC)材料相比,Click TE-GSH富集多磷酸化肽的选择性更好. 本工作所建立的富集方法为高效富集多磷酸化肽提供新方法和新技术.     

固相萃取法对微量药物的选择性富集和直接测定

选用能够识别抗菌药物氧苄氨嘧啶（ＴＭＰ）的模权聚合物作固相萃取剂,从稀溶液中可选择性地富集ＴＭＰ,在生理浓度下,富集因数为３５．７,而非模板事物无富集能力。在洗脱过程中直接用于ＴＭＰ的测定,不需要随后的色谱分析手续。基于此,建立了一个样品富集和测定的新方法,方便简单可靠,用于尿样中低２的ＴＭＰ的测定,回收率达到９１．７％,结果满意。     

药物扑热息痛分子模板聚合物的选择性富集与识别特性研究

采用分子印迹技术合成了对药物扑热息痛有高选择性的模板聚合物。通过Ｓｃａｔｃｈａｒｄ分析研究了模板聚合物的结合特性;研究了模板聚合物的结合动力学。结果表明,以丙烯酰胺为功能单体的模板聚合物比以甲基丙烯酸为功能单体的聚合物具有更高的结合容量。     

Coherent Enantiomer‐Selective Population Enrichment Using Tailored Microwave Fields

We report the experimental demonstration of coherent enantiomer‐selective enrichment of chiral molecules by employing a novel microwave five‐pulse scheme. Our results show that enantiomers can be selectively transferred to a rotational level of choice by applying sequences of resonant microwave pulses in a phase‐ and polarization‐controlled manner. This is achieved by simultaneously exciting all three kinds of electric dipole‐allowed rotational transitions and monitoring the effect on a fourth rotational transition of choice. Using molecular beams, we apply our method to two chiral terpenes and obtain a 6 % enantiomeric enrichment, which is one order of magnitude larger than that recently reported in a buffer‐gas cell experiment. This approach establishes a robust scheme for controlled manipulation of enantiomers using tailored microwave fields and opens up new avenues for chiral purification and enrichment that can be used in a broad scope of analytical or spectroscopic applications.     

铜在金电极表面的选择性化学镀富集的研究

在具有催化还原活性的金电极表面,以水合肼为还原剂,在pH10.5酒石酸钾钠溶液中,通过化学镀方法,选择性地在金电极表面沉积了单层结构的铜膜。用开路电位时间谱技术(Op～t)、循环伏安法(CV)和微分脉冲伏安法(DPV)表征了该溶液还原法对铜进行选择性富集的机理和效果。证明在多种金属离子共存的复杂溶液体系中,可以避免其它离子的干扰,使铜选择性地富集到金电极表面。化学镀浴中富集到金电极表面的单层铜膜溶出电流与Cu2+的浓度在3×10-6～1×10-4mol/L范围内呈线性关系。该法已用于矿样中铜的还原富集、分离和测定,分析结果与电感耦合等离子体发射光谱法(ICP/AES)作了比较,结果满意。     

Selective Enrichment of a Methanol-Utilizing Consortium Using Pulp and Paper Mill Waste Streams

Efficient utilization of carbon inputs is critical to the economic viability of the current forest products sector. Input carbon losses occur in various locations within a pulp mill, including losses as volatile organics and wastewater. Opportunities exist to capture this carbon in the form of value-added products such as biodegradable polymers. Waste-activated sludge from a pulp mill wastewater facility was enriched for 80 days for a methanol-utilizing consortium with the goal of using this consortium to produce biopolymers from methanol-rich pulp mill waste streams. Five enrichment conditions were utilized: three high-methanol streams from the kraft mill foul condensate system, one methanol-amended stream from the mill wastewater plant, and one methanol-only enrichment. Enrichment reactors were operated aerobically in sequencing batch mode at neutral pH and 25°C with a hydraulic residence time and a solids retention time of 4 days. Non-enriched waste activated sludge did not consume methanol or reduce chemical oxygen demand. With enrichment, however, the chemical oxygen demand reduction over 24-h feed/decant cycles ranged from 79 to 89%, and methanol concentrations dropped below method detection limits. Neither the non-enriched waste-activated sludge nor any of the enrichment cultures accumulated polyhydroxyalkanoates (PHAs) under conditions of nitrogen sufficiency. Similarly, the non-enriched waste activated sludge did not accumulate PHAs under nitrogen-limited conditions. By contrast, enriched cultures accumulated PHAs to nearly 14% on a dry weight basis under nitrogen-limited conditions. This indicates that selectively enriched pulp mill waste activated sludge can serve as an inoculum for PHA production from methanol-rich pulp mill effluents.     

Borated Titania,a New Option for the Selective Enrichment of cis‐Diol Biomolecules

As low abundance cis‐diol biomolecules are of great significance in biological organisms, preparation of materials for the selective enrichment of such compounds is highly favorable for the development of the related proteomics and metabolomics. To this end, we have prepared monolithic borated titania by a non‐aqueous sol‐gel strategy as a new inorganic affinity material for the specific capture of nucleosides, glycopeptides and glycoproteins. Benefiting from the inorganic framework, this material prevented the hydrophobic interference, which was somewhat inevitable for the mainstream organic‐based boronate affinity materials. The prepared material was carefully characterized by scanning electron microscope (SEM), X‐ray diffraction (XRD), X‐ray photoelectron spectroscopy (XPS) and nitrogen‐sorption experiments to investigate the morphology and elemental composition. The excellent performance of borated titania on enrichment of cis‐diol biomolecules was demonstrated by extracting the glycopeptides from horseradish peroxidase (HRP) digestion, standard glycoproteins, and nucleosides from a human‐urine matrix. This kind of inorganic affinity material offers a new option for selective enrichment or separation of cis‐diol biomolecules.     

Metal-Loaded Sorbents for Selective On-Line Sample Handling and Trace Enrichment in Liquid Chromatography

Abstract

Three different commercially available stationary phases containing a thiol, an 8-hydroxyquinoline (oxine), and a 2-amino-1-cyclopentene-1-dithiocarboxylic acid (ACDA) functional group, were loaded with mercury (Hg(II)), platinum (Pt(IV)) and silver (Ag(I)) ions. The phases were packed in small precolumns and evaluated for their potential towards the selective on-line sample handling and trace enrichment of three model systems in liquid chromatography. The thiol 2-mercaptobenzimidazole was used to study trace enrichment on Hg(II)-loaded phases; the herbicide buturon, which contains an ethynic bond, was selected to study trace enrichment on Ag(I)-loaded phases. The on-line filter effect of Pt(IV)-loaded phases was investigated with 4-chloroaniline as a model compound.

The results indicate that the Hg(II)-ACDA phase should be preferred for the trace enrichment of thiols, a Pt(IV)-ACDA or a Pt(IV)-thiol phase for the anilines and the Ag(I)-oxine phase for the trace enrichment of ethynic compounds.

As environmental application, the selective on-line trace enrichment of buturon from river water samples, on a precolumn packed with Ag(I)-oxine, is shown.     

聚多巴胺修饰棉花固定金属离子用于磷酸化多肽的富集

本文通过多巴胺自聚合在天然的棉花纤维表面,构建了仿生聚多巴胺(PDA)膜层,然后利用儿茶酚羟基固定Ti~(4+),设计并合成了一种固定金属亲合色谱(Immobilized Metal Ion Affinity Chromatography,IMAC)材料Cotton@PDA-Ti~(4+),并将其用于磷酸化多肽的富集。该材料机械性能好,化学性能稳定和生物相容性好,且制备过程简单,通过简易的In-pipet-tip固相萃取(SPE)装置使整个富集操作过程简便快速。实验结果表明,Cotton@PDA-Ti~(4+)不仅可以从简单的蛋白酶解物(β-casein)中富集磷酸化多肽,并且在含有大量非磷酸化多肽的复杂体系样品中对磷酸化多肽也表现出良好的选择性。另外,利用Cotton@PDA-Ti~(4+)对磷酸化多肽进行富集也有较高的效率。我们将该材料应用于实际样品,如人体血清以及脱脂牛奶酶解物中磷酸化多肽的富集,均表现出了较好的选择性。说明该方法有可能用于磷酸化蛋白质组的全分析。     

基于激光介导富集的表面增强拉曼分析

基于具有优异表面增强拉曼散射(SERS)性能的石墨烯隔离的金纳米晶(GIAN)能够在水-有机相界面自组装, 待测物分子在有机相中的分配系数较大以及GIAN能够通过π?π相互作用与待测物分子结合的优势, 构建了激光介导的待测物分子的高效富集策略, 进而实现了9,10-双苯乙炔基蒽(BPEA)分子的痕量SERS分析. 所构建的新型待测物分子高效富集策略在一定程度上避免了因“咖啡环效应”带来的信号波动, 有望为复杂体系中痕量待测物的SERS分析提供可靠的平台.     

基于离心式富集装置的酪氨酸磷酸肽分析新方法

酪氨酸磷酸化及其相应激酶活性的研究在抗肿瘤药物靶点的研发中具有重要意义.由于酪氨酸磷酸化仅占蛋白质总磷酸化含量的不足0.1%,因此规模化的酪氨酸磷酸化鉴定面临着重大技术挑战.本研究构建了TiO2串联C18反相填料的离心式富集装置,结合抗体免疫沉淀法,建立了酪氨酸磷酸肽的富集策略.此新型富集装置由吸头、适配器和离心(EP)管组成,将TiO2富集磷酸肽和C18填料反相分离磷酸肽有机结合,以离心的方式进行样品的上样、清洗、洗脱和分离,再通过抗酪氨酸磷酸化抗体进一步特异性富集酪氨酸磷酸肽,从而实现了酪氨酸磷酸肽的高效富集和大规模质谱鉴定.通过离心式富集装置简化了实验步骤,减少了样品损失和人为因素干扰;而且离心式、平行化的样品处理方式可显著提高分析通量.将此策略成功用于小鼠肝脏蛋白质酪氨酸磷酸化肽段的富集和质谱鉴定,在5 mg鼠肝蛋白中共鉴定出967个酪氨酸磷酸化位点,对应545个蛋白质,显示了其在蛋白质组学研究中的应用潜力.     

水中丙溴磷的富集及气相色谱分析

采用ＧＤＸ－５０１吸附剂富集水中痕量丙溴磷,然后用气相色谱法直接测定,方法简便、准确,实用。水中丙溴磷的检出限为０．４μｇ／Ｌ,低、中、高３种浓度的平均加标回收率为８８．６％－９９．３％,相对标准偏差在３．２３％－４．９６％范围内。     

X射线荧光光谱分析不锈钢时的数学校正方法

本文探讨了用XRF法分析不锈钢时，用个别交叉项系数校正Cr、Fe、Ni之间的交叉效应，从而有效地修正了它们之间的吸收一增强效应，使分析结果得到改善，最大误差不超过0．5％。与C－Q方程相比，该法计算简便，具灵活性和实用性，可推广使用。