Aerolysin纳米孔道对寡聚核苷酸的高灵敏单分子检测

自纳米孔道单分子电化学技术提出以来,为了构建性能良好的纳米孔道,研究人员一直在寻找不同的孔道材料. 本研究探索了Aerolysin生物纳米孔道在寡聚核苷酸检测方面的可能性. 实验结果表明,与常用的α-溶血素纳米孔道相比,Aerolysin纳米孔道在寡聚核苷酸检测方面表现出更强的空间和时间分辨能力. 三个碱基长度的寡聚核苷酸可对Aerolysin纳米孔道造成约为40%的电流阻断. 阻断时间表现出电压相关性,随电压的升高而减小. 与其他生物纳米孔道相比,Aerolysin纳米孔道无需任何基因突变、化学修饰即可实现对单个寡聚核苷酸的超灵敏分析. 未来,Aerolysin纳米孔道将有可能应用于DNA损伤检测、microRNA分析以及其他基于纳米孔道的单分子分析检测.     

Aerolysin蛋白纳米孔道直接检测单个DNA碱基修饰

基于Aerolysin生物膜通道蛋白的纳米孔道电化学分析技术,因其高的电化学空间限域能力可实现超灵敏DNA单分子检测。本文利用单个Aerolysin纳米孔道在无需标记、无需扩增的条件下直接分辨3种具有单个碱基差异的单链DNA。实验结果显示,具有单个炔基侧链基团修饰的单个ss DNA在限域空间内与Aerol-ysin纳米孔道的相互作用时间较未修饰的ss DNA增长近7倍,电流阻断程度增大7%,且高斯峰半峰宽减小了44%,增强了Aerolysin纳米孔道对单个DNA分子的分辨能力。研究成果有望推动Aerolysin纳米孔在DNA直接测序及表观遗传修饰检测中的应用。     

基于Aerolysin纳米孔道对单个c-di-AMP分子的检测研究

环二腺苷酸(c-di-AMP)是原核细胞中普遍存在的第二信使, 不仅能够有效调控细胞生长、离子转运、细胞壁代谢平衡等多种生理过程, 还能引发I型干扰素应答, 激发机体天然免疫反应. 本实验使用单个气单胞菌溶素(Aerolysin)纳米孔道蛋白构建的单分子界面, 对c-di-AMP进行单分子测量研究. 为提高Aerolysin纳米孔对带负电小分子化合物的测量灵敏度, 本实验利用LiCl为支持电解质, 有效屏蔽Aerolysin孔口表面负电荷, 减小c-di-AMP与Aerolysin纳米孔之间的静电排斥, 从而显著增强了Aerolysin纳米孔道对单个c-di-AMP分子的检测能力. 实验结果显示, 在90 mV电压下, 每分钟在LiCl中获得的有效穿孔事件的数量最高可达同条件KCl支持电解质的30倍, 且有效穿孔事件占总体事件的比例在不同电压下提升了7~11倍. 进一步表明, 使用LiCl支持电解质, 可有效增强Aerolysin孔道对带负电小分子化合物的测量灵敏度. 因此, 本研究实现了Aerolysin纳米孔道对单个环二核苷酸的高灵敏免标记检测, 有望为单分子水平上阐明新型免疫干扰机制提供新的分析方法.     

单分子电泳:纳米孔道电化学的新认识

该文旨在从电泳分离技术的角度认识纳米孔道电化学单分子分析技术,这种技术可以作为"单分子电泳"来理解和研究。纳米孔道电化学单分子分析技术与电泳的本质都是采用外加电场使待测分子产生电迁移。待测分子性质不同,且与介质材料孔道外露基团相互作用不同,使得分子移动速度具有差异,据此实现分离识别。气单胞菌溶素（Aerolysin）纳米孔道,由于其孔径与待测分子尺寸相匹配,其孔道内壁可以看作是由氨基酸组成的具有调控单个分子电迁移能力的特异性孔道界面。每一个氨基酸残基都相当于一个探测单元,在电场力的作用下,待测分子逐一进入孔道时与每一个探测单元相互作用方式、程度与时长不同,从而形成了单个待测分子特征的迁移速度和迁移运动轨迹。在纳米孔道实验中,每秒可以有上千个待测分子穿过孔道,产生特征阻断电流信号。通过对这些信号的阻断电流、阻断时间、阻断频率、信号特征等进行统计分析,可以从"单分子电泳"水平对单个待测物实现高通量的分辨和识别。该文以Aerolysin纳米孔道分辨仅有一个核苷酸差异的寡聚核苷酸（5'-CAA-3'、5'-CAAA-3'、5'-CAAAA-3'）为例,详细阐述了纳米孔道"单分子电泳"的单核苷酸分辨能力,展现了电化学限域空间在电泳单分子水平分离技术上的应用。     

纳米孔道单分子电化学测量的低噪音控温系统研究

纳米孔道检测技术是一种利用单个分子测量界面实现在单分子水平上测量DNA、RNA、蛋白、多肽等生物分子的高灵敏的单分子检测技术. 由于单个分子与纳米孔道的相互作用受热力学控制,亟需精准控制纳米孔道单分子分析的实验温度. 因此,本文研制了一种低噪音控温系统用于具有皮安级电流分辨的纳米孔道单分子实验,以实现精确调控测量时的环境温度. 该系统利用半导体制冷片的热电效应对检测池环境加热/制冷,通过对高精度热敏电阻进行电磁屏蔽以实现在温度反馈的同时避免噪音的引入. 利用比例-积分-微分算法进行控制,达到高精度快速控温的要求. 该系统控温精度为±1 °C,无额外噪音引入至超灵敏纳米孔道单分子测量,获得了25 °C到5 °C下Poly(dA)5与单个气单胞菌溶素（Aerolysin）分子界面间作用产生信号的差异,应用于研究单分子与纳米孔道相互作用的热力学行为.     

Aerolysin单分子界面的构建及高选择性单分子检测

提出了一种基于Aerolysin膜蛋白质分子构建单分子界面的方法, 运用蛋白质工程技术对单分子界面进行定点修饰, 所建立方法灵活、 可控且重复性好. 采用Poly(dA)₄为探针分子对修饰后的单分子界面进行了表征, 结果表明, 在孔口处的Arginine修饰影响了寡聚核苷酸的选择性. 为进一步理解Aerolysin单分子界面及合理设计功能性单分子界面提供了参考.     

纳米通道单分子检测P53蛋白与DNA的弱相互作用

利用基于α-溶血素(α-HL)的纳米通道单分子检测技术在单分子水平上探测位于肿瘤抑制蛋白P53 DNA结合域的多肽分子(P53-P)与含有p21^waf1/cip1基因结合域的双链DNA (B40)之间弱相互作用. 在电场力的作用下, 单个P53-P/B40复合物被α-HL纳米通道捕获并限制在其前庭中. 通过空间限域作用, P53-P与B40之间的弱相互作用被放大为具有显著电压依赖性的复合物-α-HL间强烈的相互作用, 从而产生极易分辨的离子流特征阻断信号. 统计分析特征阻断信号显示P53-P与B40之间的弱相互作用使得B40产生微小的构型变化. 分子对接模拟进一步证明, P53-P能够插入B40的小沟并使得小沟间距变窄. 因此, 基于α-HL的纳米通道单分子检测技术可以作为研究单个生物大分子间弱相互作用的超灵敏分析方法.     

柔红霉素与DNA作用的序列特异性研究

采用紫外-可见光谱法和紫外-可见光谱电化学法研究了柔红霉素(DNR)与不同寡聚核苷酸之间的相互作用.结果表明,DNR优先作用于寡聚核苷酸的CpG位点,然后是ApG和ApC.因为DNR可与鸟嘌呤之间形成3个氢键.与双链寡聚核苷酸作用时,DNR最先插入的位点是(CpG)₂碱基对之间,其次是(TpG)(CpA)和(GpC)(ApC)碱基对之间.当DNR与存在未配对G碱基的DNA链作用时,因游离的DNR量增加使其电化学活性增加,导致DNA构象和构型的变化,使DNA生理功能受到损伤,DNA碱基增色效应显著上升.此法可用于G碱基未配对DNA链的检测.     

盐酸小檗碱与DNA作用的序列特异性研究

基于分子吸收和发射测量,研究了盐酸小檗碱与特定序列DNA间的相互作用.结果发现双链DNA中含有不配对 G 碱基时,其与盐酸小檗碱作用后产生的光谱特征将明显不同于完全互补的双链DNA.该特征可被用于检测1个错配碱基的双链DNA.利用 Hyperchem Professional 软件包计算了4种寡聚核苷酸的电荷分布情况,并在此基础上进一步探讨了盐酸小檗碱与寡聚核苷酸之间的作用机理.     

用于纳米孔道分析的四通道电化学传感器设计与仿真

纳米孔道技术是一种基于空间限域的超灵敏的单分子分析技术.通过研究单个分子限域于纳米孔道中所产生的离子电流的变化,可在单分子尺度上获取其结构、尺寸、电性及与孔道间弱相互作用的信息.目前主要应用的纳米孔道测量仪器单次实验仅能测量单个纳米孔道,其检测通量较低.本文基于实验室前期自主设计研制的单通道纳米孔道测量仪器Cube-D2上,比较研究了两种互阻放大器的测量特性,从而选择了合适的测量电路设计了四通道电化学传感器放大电路.进一步通过仿真验证了四通道电化学传感器设计方案的可行性,为阵列化高通量纳米孔道单分子电化学测量仪器的设计提供了理论基础.     

二氧化碳和丙烯直接合成甲基丙烯酸的NiPMo/TiO2催化剂的研究

用溶胶-凝胶法以磷钼酸(MPA)的镍盐溶液水解钛酸四丁酯制备了NiPMo/TiO2催化剂.使用ICP、 XRD、 TG-DTA、 IR、 TPD-MS和微反应技术研究了催化剂的化学组成、热稳定性、化学吸附性质和催化反应性能.杂多钼酸盐与TiO2通过O2-在TiO2表面发生了键合.在623 K下,杂多阴离子仍保持原有的Keggin结构.CO2在Lewis酸位Ni(Ⅱ)和Lewis碱位Ni-O-Mo的桥氧协同作用下生成CO2卧式吸附态Ni(Ⅱ)←O-(CO)←(O--Ni).丙烯有多种吸附态在催化剂上吸附.在563 K、 1 MPa和空速1500 h-1的反应条件下,丙烯的摩尔转化率为3.2%,产物MAA选择性为95%.     

α-Amino acid derived enaminones and their application in the synthesis of N-protected methyl 5-substituted-4-hydroxypyrrole-3-carboxylates and other heterocycles

A new and simple synthesis of novel N-protected methyl 5-substituted-4-hydroxypyrrole-3-carboxylates, which exist in equilibrium with their 4-oxo tautomers, has been developed in two steps starting from N-protected α-amino acids. The key intermediates are enaminones, which can also be isolated, characterized, and used for the construction of other functionalized heterocycles, before they spontaneously decompose to pyrrole products. 4-Hydroxypyrroles are prone to partial aerial oxidation but can be efficiently alkylated or reduced to stable polysubstituted pyrrolidine derivatives.     

Specific intramolecular aromatic CH insertion of diazosulfonamides

The chemoselectivity in the intramolecular CH insertion of various diazosulfonamides has been experimentally studied. The results reveal that the aliphatic 1,4-, 1,5-, or 1,6-C(sp³)?H insertions of diazosulfonamides are not accessible, while the aromatic 1,5-C(sp²)?H insertion can be realized specifically by adjusting the diazo-adjacent group. In addition, the general chemoselectivities in the intramolecular CH insertions of diazosulfonyl compounds are summarized. Generally, diazosulfones undergo both aromatic 1,5-C(sp²)?H and aliphatic 1,5- and 1,6-C(sp³)?H insertions, while diazosulfonates undergo aliphatic 1,5- and 1,6-C(sp³)?H insertions. However, diazosulfonamides only undergo aromatic 1,5-C(sp²)?H insertion.     

CoFe2O4自形成磁性液体场致结构化对磁化的影响

The Langevin paramagnetic theory can’t describe the relation between magnetization of ferrofluids and applied magnetic field. The structuralization of ferrofluids, which is considered the main influence factor of the magnetization, is regarded. The part of magnetization works is deposited when the structure is forming. This action influences the magnetization of ferrofluids directly or indirectly. On the base of the “compressing” model, the Langevin function that usually describes the magnetization of ferrofluid is modified, and a well-fitted curve is obtained. An equation of the relation between the equivalent volume fraction after being “compressed” and the intensity of magnetic field is discovered, which approximately describes the process of magnetization. The relation between the approximate initial susceptibility and the volume fraction can be obtained from modified formula.     

Highly regioselective Buchwald–Hartwig amination at C-2 of 2,4-dichloropyridine enabling a novel approach to 2,4-bisanilinopyridine (BAPyd) libraries

The highly regioselective Buchwald–Hartwig amination at C-2 of the cheap and readily accessible reagent, 2,4-dichloropyridine with a range of anilines and heterocyclic amines is described. This new methodology is robust and provides a facile access to 4-chloro-N-phenylpyridin-2-amines on 0.25 mol scale. These intermediates undergo a further Buchwald–Hartwig amination at higher temperature to enable rapid exploration of the chemical space at C-4 and to provide a library of 2,4-bisaminopyridines.     

N-Heterocyclic carbene-palladacyclic complexes: synthesis,characterization and their applications in the C-N coupling and α-arylation of ketones using aryl chlorides

N-Heterocyclic carbene-palladacyclic complexes 3 were successfully achieved in a one-pot procedure under mild conditions. The structure of 3a was unambiguously confirmed by X-ray single crystal diffraction and it was an active catalyst in the Buchwald-Hartwig amination and α-arylation of ketones even at very low catalyst loadings (0.01?mol%).     

Iodine-mediated oxidative Pictet-Spengler reaction using terminal alkyne as the 2-oxoaldehyde surrogate for the synthesis of 1-aroyl-β-carbolines and fused-nitrogen heterocycles

An efficient iodine-mediated oxidative Pictet-Spengler reaction in dimethyl sulphoxide (DMSO) using terminal alkynes as the 2-oxoaldehyde surrogate for the synthesis of aryl (9H-pyrido[3,4-b]indol-1-yl)methanones is described. The scope of the protocol includes the total synthesis of Fascaplysin, Eudistomins Y₁ and Y₂. The methodology is extended for preparing pyrrolo[1,2-a]-quinoxaline and indolo[1,5-a]quinoxaline derivatives. The utility of 1-aroyl-β-carbolines was demonstrated by performing palladium-catalyzed β-carboline directed ortho-C(sp2)-H functionalization of the phenyl ring with thiomethyl (SMe) group using DMSO as source and for accessing 4-aryl-canthin-6-ones.     

Facile synthesis of bicyclic 1-arylpyrazol-5-ones

In this Letter, we described a facile method for constructing fused bicyclic 1-arylpyrazol-5-one ring system. We employed various methylene-containing carboxylic acids as the substrates and proved that the pyrazolone ring closure requires activated methylene group in intermediate II. Accordingly, a series of structurally diversified, fused bicyclic 1-arylpyrazol-5-ones was prepared in moderate to high yields using the requisite substrates.     

Sulfur-directed metal-free and regiospecific methyl C(sp3)–H imidation of thioanisoles

Regiospecific and direct imidation of the methyl C(sp³)–H bond of thioanisoles is realized under mild and metal-free conditions with N-fluorobis(benzenesulfonyl)imide as an oxidant and nitrogen source. Proposed mechanism suggests that thionium ion intermediates and a Pummerer-type reaction are involved. The imidation has advantages such as high step-economy, excellent functionality tolerance, and regiospecificity, giving structurally diverse imidation products.