红外光谱法研究重氮树脂自组装膜的光化学反应

将具有导电功能的聚(4-羧酸钠苯基)乙炔阴离子(PCPA)与具有生物活性的DNA聚阴离子交替与重氮树脂聚阳离子(DR)进行自组装,可以得到层重复单元为DR／DNA／DR／PCPA的三夹层聚电解质自组装膜。重氮树脂聚阳离子在紫外光照射下可与聚阴离子发生光化学反应,使层与层之间的离子键连接转化为共价键连接,从而制备稳定的纳米级超薄膜。本文用红外光谱法对此类聚电解质自组装膜的光化学反应过程进行了研究。     

有一未配对残基T的DNA [d(TCGCGCG)]_2结构特征的NMR研究

具有交替的嘧啶-嘌呤序列和5′端有一未配对残基T的DNA七聚体[d(TCGCGCG)]_2可以作为研究DNA双螺旋的双链-单链联结构象特征的模本。DNA的这种联结方式存在于它们的复制和转录过程中。用NOESY和TOCSY/HOHAHA实验对其共振峰作了指定。对七个不同混合时间的NOE强度进行了定量分析,用双自旋近似法计算了质子间的距离,揭示了这个分子中令人感兴趣的结构特征。用P.E.COSY实验测定了该DNA双螺旋各糖质子间偶合常数,鉴定了各糖的构象。从而获得了该DNA七聚体在溶液中的结构特征。就整体结构而言,该DNA分子仍属B族DNA,但结构有了很大变化,特别是在端基区。未配对的残基具有不同寻常的顺式构象。为DNA双链-单链联结部分构象提供了有用的借鉴。     

聚谷氨酸为骨架的梳状基因载体的合成及生物学性能评价

以聚谷氨酸为骨架, 用低分子量聚乙烯亚胺胺解聚谷氨酸苄酯, 得到聚谷氨酸-g-聚乙烯亚胺, 用异佛尔酮二异氰酸酯将聚乙二醇单甲醚偶联到聚谷氨酸-g-聚乙烯亚胺上, 合成了梳状聚阳离子基因载体聚谷氨酸-g-(聚乙烯亚胺-b-聚乙二醇). 利用核磁共振氢谱、 激光粒度分析仪、 Zeta电位仪和凝胶电泳对聚阳离子载体及其与质粒脱氧核糖核酸(pDNA)形成的复合物进行了表征. 通过噻唑蓝(MTT)细胞毒性测试、 绿色荧光蛋白质粒pEGFP-C1及荧光素酶质粒pGL3体外转染实验考察了载体的细胞毒性及基因转染效率. 结果表明, 当聚乙烯亚胺中N原子和DNA中P原子的摩尔比(N/P)大于5时, 载体能很好地包裹DNA, 载体与DNA形成的复合物粒径约为130 nm, Zeta电位约为28 mV; 通过MTT实验和体外质粒转染实验显示出载体在测量范围内具有极低的细胞毒性和较高的转染效率.     

负载肝素及生物素化肝素修饰的纳米粒子的基质介导基因传递系统

首先通过静电作用将带负电荷的肝素及生物素化肝素和带正电荷的PAMAM/DNA自组装,分别制备了PAM AM/DNA/heparin和PAMA M/DNA/heparin-biotin复合物,然后用快速降解胆酸功能化星型聚(DL-丙交酯)作为基质、并加入水溶性高分子α,β-聚(N-2-羟乙基)-L-天冬酰胺(PHEA)作...     

聚多巴胺包埋G-四联体/血红素DNA酶制备过氧化氢生物传感器

利用多巴胺的氧化自聚实现对G-四联体/血红素DNA酶的包埋,成功构建了H2O2电化学生物传感器。DNA和血红素混合得到G-四联体/血红素复合物;DNA酶物理吸附在玻碳电极上后,将10μL 5 g/L多巴胺的磷酸盐缓冲液(pH 8.0)滴在表面,空气中的氧气氧化多巴胺形成聚多巴胺膜,实现DNA酶的固定。考察了不同DNA序列对传感器性能的影响,表明电化学与光学传感过程具有不同序列响应。此传感器对H2O2的检出限为2.2μmol/L;线性范围为0.01～1.5 mmol/L。本研究证实了利用聚多巴胺固定酶和用DNA酶代替天然酶构筑传感器的可行性。     

PLL-PEG-PLL三嵌段共聚物的制备及DNA包覆应用

将PLL(Z)-PEG-PLL(Z)中的氨基去保护得到两端为聚阳离子的嵌段共聚物PLL-PEG- PLL, 并对与DNA结合形成的聚集体的性质进行了研究. 结果表明: PLL-PEG-PLL与DNA在水相中形成了聚离子复合物胶束; DNA与PLL通过静电作用形成核, PEG包裹在外面, 得到的聚离子复合物胶束为均一体系, 即使在化学计量点也不会沉淀, 保持着很好的流动性和稳定性, 这一点对基因治疗是很重要的; 而且随着PLL段的增加, 包覆效果更好; 聚离子复合物胶束使得DNA具有较强的核酶抗性; 大小在10~35 nm之间, 主要呈球形; 聚离子复合物胶束能够用于基因的传递, 使基因在细胞系SF9中表达出蛋白质.     

高效毛细管电泳用于以聚环氧乙烷为筛分介质分离DNA的机理研究

用毛细管电泳以聚环氧乙烷(PEO)为筛分介质对pUC19DNA/Msp Ⅰ(HpaⅡ)Marker中的12条DNA片段进行了分离,并尝试用Ogston模型、爬行模型以及线性模型对分离机理进行研究,最终发现26～147bp的小片段,在低电场强度时能很好地符合Ogston模型理论,而190～501bp中等长度的DNA片段电泳迁移率与其尺寸间存在很好的负相关的线性关系,为此,提出一种新的线性模型来进行解释.此外,还探讨了PEO的浓度和电场强度对分离的影响.其结论可更好地从理论上指导对中小片段DNA的分离,对肿瘤基因突变点的分析和PCR扩增产物的分离分析具有重要的意义.     

基于阳离子共轭聚合物比色检测HIV逆转录酶的RNase H活性

利用阳离子聚噻吩衍生物与单链DNA和杂合体DNA/RNA通过静电相结合时所产生的紫外吸收变化,建立了一种检测HIV逆转录酶(RT-HIV)的RNase H活性的方法。阳离子聚噻吩衍生物的紫外吸收最大波长位于短波385nm,与单链DNA结合会使聚噻吩衍生物的紫外吸收最大波长红移至525nm;而与杂合体DNA/RNA结合时对其紫外吸收最大波长几乎没有影响,当利用RT-HIV的核糖核酸酶RNase H活性水解掉杂合体中的RNA时,杂合体溶液又会使聚噻吩衍生物的紫外吸收最大波长发生红移。结果表明,紫外吸收最大波长变化明显,甚至直观用肉眼就可以观察到杂合体水解前后溶液颜色的变化。同时还测定了不同时间下RNase H酶水解杂合体中RNA的吸光度变化曲线,计算出了RNase H酶水解的动力学常数和最大初速度。     

核苷酸、聚核苷酸和核酸猝灭Tb~(3+)-钛铁试剂络合物荧光的机理研究

研究了核苷酸、聚核苷酸和核酸对Tb3+－钛铁试剂（TR）络合物的荧光碎灭机理，认为荧光猝灭过程是核苷酸、聚核苷酸和核酸分子中的磷酸基组分与TR竞争Tb3+离子，生成实验条件下无荧光的二元络合物的静态猝灭过程；用Tb3+－TR络合物荧光探针研究DNA嵌入剂和金属离子与DNA相互作用的实验结果说明这一机理是合理的．     

CMC-g-DNA接枝共聚物的合成与表征

在磷酸盐缓冲溶液中用1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)与N-羟基琥珀酰亚胺(NHS)活化羧甲基纤维素钠(CMC)侧链上的羧基; 在室温下再将活化的CMC与5'端经氨基修饰的单链脱氧核糖核酸(DNA)齐聚物(ODNs)反应, 获得CMC上接枝ODNs的共聚物(CMC-g-ODNs), 以Lambda DNA为模板, 通过聚合酶链式反应(PCR), 将接枝的ODNs扩增为长度为1300个碱基对的双链DNA, 从而制得CMC侧链上接枝DNA的共聚物CMC-g-DNA. 采用傅里叶红外光谱仪测定CMC与NHS形成的中间体; 用水平式琼脂糖凝胶电泳和垂直板变性聚丙烯酰胺凝胶电泳对CMC-g-DNA接枝共聚物进行表征. 结果表明, 合成了CMC-g-DNA接枝共聚物, 且在酸性条件下CMC的活化效果更好; 同时, 接枝在CMC上的DNA在琼脂糖凝胶电泳中迁移速率加快, 而在聚丙烯酰胺凝胶电泳中迁移速率减慢.     

Direct Electrochemistry of Hemoglobin in Layer‐by‐Layer Films Assembled with DNA and Room Temperature Ionic Liquid

Based on electrostatic interaction and electrodeposition, poly‐anionic deoxyribonucleic acid (DNA), room temperature ionic liquid 1‐butyl‐3‐methyl‐imidazolium tetrafluoroborate (BMIMBF₄), hemoglobin (Hb) and Poly(diallyldimethylammonium chloride) (PDDA) were successfully assembled into Hb/IL/DNA/PDDA layer‐by‐layer complex films on the surface of ITO electrode. FTIR spectroscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the composite film. The obtained results demonstrated that the Hb molecule in the film kept its native structure and showed its good electrochemical behavior. A pair of well‐defined redox peaks of Hb with the formal potentials (E°′) of ?0.180 V (vs. SCE) was appeared in phosphate buffer solution (PBS, pH 7.0). The Hb/IL/DNA/PDDA/ITO modified electrode also showed an excellent electrocatalytic behavior to the reduction of hydrogen peroxide (H₂O₂). Therefore, the IL/DNA/PDDA complex film as a novel matrix open up a possibility for further study on the direct electrochemistry of other proteins and the fabrication of the third‐generation electrochemical biosensors.     

Adsorption and Electrooxidation of DNA at Glassy Carbon Electrodes Modified with Multiwall Carbon Nanotubes Dispersed in Glucose Oxidase

We are proposing for the first time the successful immobilization of DNA at glassy carbon electrodes (GCE) modified with carbon nanotubes (CNT) dispersed in glucose oxidase (GOx) (GCE/CNT‐GOx) either by direct adsorption or by layer‐by‐layer self‐assembling using polydiallyldimethylamine (PDDA). The presence of GOx allows an efficient dispersion of CNT and gives a most favorable environment that promotes the adsorption and makes possible a more sensitive electrooxidation of DNA. The PDDA incorporated in the self‐assembled architecture largely facilitates the adsorption and electrooxidation of dsDNA and the adsorbed layer can be successfully used for evaluating the interaction of DNA with methylene blue.     

Preparation of a Reduced Graphene Oxide Wrapped Lithium‐Rich Cathode Material by Self‐Assembly

A lithium‐rich cathode material wrapped in sheets of reduced graphene oxide (RGO) and functionalized with polydiallyldimethylammonium chloride (PDDA) was prepared by self‐assembly induced from the electrostatic interaction between PDDA–RGO and the Li‐rich cathode material. At current densities of 1000 and 2000 mA g^?1, the PDDA–RGO sheet wrapped samples demonstrated increased discharge capacities, increasing from 125 to 155 mA h g^?1 and from 82 to 124 mA h g^?1, respectively. The decreased resistance implied by this result was confirmed from electrochemical impedance spectroscopy results, wherein the charge‐transfer resistance of the pristine sample decreased after wrapping with the PDDA–RGO sheets. The PDDA–RGO sheets served as a protective layer sand as a conductive material, which resulted in an improvement in the retention capacity from 56 to 81 % after 90 cycles.     

血清样品中乙肝病毒的DNA电化学传感器检测

利用自组装单分子膜技术，将巯己基修饰的具有乙肝病毒(HBV)DNA序列特异性的单链DNA探针固定在金电极表面，制得DNA电化学传感器；以电活性的Hoechst 33258为指示剂，考察了该传感器对血清样品中乙肝病毒DNA的响应；探讨DNA电化学传感器在临床检测中的应用；将传感器法与荧光聚合酶链反应(PCR)法进行对比，两者的分析结果具有一致性。     

毛细管电泳-修饰电极电化学检测法测定饮用水中溴酸盐

通过静电组装技术在碳圆盘电极（PGE）表面制备{聚二烯丙基二甲基氯化铵（PDDA）/多壁碳纳米管（MWCNT）}n/PDDA多膜,并采用循环伏安法在多膜表面电化学修饰一磷钼酸（PMo12）膜,构筑PGE/{PDDA/MWNTs}5/PDDA/PMo12复合膜修饰电极,研究该复合膜修饰电极电化学及其对溴酸盐（BrO3-）电催化还原性质.在此基础上建立毛细管电泳-PGE/{PDDA/MWNTs}5/PDDA/PMo12修饰电极电化学检法定饮用水中溴酸盐分析新方法.在优化实验条件下,电泳峰面积与溴酸根浓度在5.0×10-8～5.0×10-5mol/L范围内呈良好性关系（r=0.9954）,检出为2.0×10-8mol/L（S/N=3）.     

Cyclic voltammetric detection of chemical DNA damage induced by styrene oxide in natural dsDNA layer-by-layer films using methylene blue as electroactive probe

In this work, an electrochemical sensor for detecting damage of natural double-stranded DNA (dsDNA) was constructed based on PSS/{PDDA/dsDNA}₃ layer-by-layer films, where PSS was poly(styrene sulfonate) and PDDA was poly(diallyldimethyl ammonium). When the PSS/{PDDA/dsDNA}₃ films assembled on pyrolytic graphite (PG) electrodes were immersed into methylene blue (MB) solution and loaded MB into the films, the formed PSS/{PDDA/dsDNA}₃-MB films in blank buffers at pH 7.0 showed a reversible cyclic voltammetric (CV) peak pair at −0.23 V vs. SCE for MB redox couple. In blank solutions, the MB loaded into the films was released gradually from the films, but the complete reloading of MB into the films could be realized by immersing the films into MB solution again, indicating the good reversibility of MB incorporation. However, after incubation in the solution of known genotoxic agent styrene oxide (SO), the damaged PSS/{PDDA/dsDNA}₃-MB films could not return to their original and fully-loaded state with reloading of MB, and showed smaller CV peak currents than those of intact PSS/{PDDA/dsDNA}₃-MB films. The relative peak current ratio, I_p,I/I_p,III, where I_p,I was the anodic peak current of intact DNA films in blank buffers after fully loading with MB and I_p,III was that of SO-damaged DNA films after reloading with MB, increased linearly with the incubation time with SO in the range of 5–40 min with a damage rate of 0.0099 min⁻¹. While the formation of SO adducts with dsDNA had no substantial effect on the dsDNA conformation, the steric hindrance of SO adducts with guanine or adenine blocked the intercalation of MB into the base pairs of dsDNA, resulting in the decrease of CV peak currents of loaded MB. The specific intercalation of MB into dsDNA base pairs and the sensitive electrochemical response of MB, combined with the unique feature of loading reversibility of MB in the DNA layer-by-layer films, make the difference in CV response between the intact and damaged dsDNA films become pronounced in the “loading/release/reloading” procedure. This may provide a new and general approach for constructing a biosensor for screening genetoxic chemicals in vitro.     

A Competitor-switched Electrochemical Sensor for Detection of DNA

A competitor‐switched electrochemical sensor based on a generic displacement strategy was designed for DNA detection. In this strategy, an unmodified single‐stranded DNA (cDNA) completely complementary to the target DNA served as the molecular recognition element, while a hairpin DNA (hDNA) labeled with a ferrocene (Fc) and a thiol group at its terminals served as both the competitor element and the probe. This electrochemical sensor was fabricated by self‐assembling a dsDNA onto a gold electrode surface. The dsDNA was pre‐formed through the hybridization of Fc‐labeled hDNA and cDNA with their part complementary sequences. Initially, the labeled ferrocene in the dsDNA was far from surface of the electrode, the electrochemical sensor exhibited a "switch‐off" mode due to unfavorable electron transfer of Fc label. However, in the presence of target DNA, cDNA was released from hDNA by target DNA, the hairpin‐open hDNA restored its original hairpin structure and the ferrocene approached onto the electrode surface, thus the electrochemical sensor exhibited a "switch‐on" mode accompanying with a change in the current response. The experimental results showed that as low as 4.4×10⁻¹⁰ mol/L target DNA could be distinguishingly detected, and this method had obvious advantages such as facile operation, low cost and reagentless procedure.     

A sensitive amperometric immunosensor for alpha-fetoprotein based on carbon nanotube/DNA/Thi/nano-Au modified glassy carbon electrode

A novel amperometric immunosensor for the determination of alpha-fetoprotein (AFP) was constructed using films of multi-wall carbon nanotubes/DNA/thionine/gold nanoparticles (nano-Au). Firstly, multi-wall carbon nanotubes (MWCNT) dispersed in poly(diallydimethlammonium chloride) (PDDA) were immobilized on the nano-Au film which was electrochemically deposited on the surface of glassy carbon electrode. Then a negatively charged DNA film was absorbed on the positively charged PDDA. Subsequently, thionine was attached to the electrode via the electrostatic interaction between thionine and the DNA. Finally, the nano-Au was retained on the thionine film for immobilization of AFP antibody (anti-AFP). The modification process was characterized by cyclic voltammetry (CV) and scanning electron microscope (SEM). The factors possibly influenced the performance of the proposed immunosensors were studied in detail. Under optimal conditions, the proposed immunosensor exhibited good electrochemical behavior to AFP in a two concentration ranges: 0.01–10.0 and 10.0–200.0 ng/mL with a relatively low detection limit of 0.04 ng/mL at three times the background noise. Moreover, the selectivity, repeatability and stability of the proposed immunosensor were acceptable.     

差分脉冲伏安法同时检测硝苯地平和尼群地平

采用绿色环保的还原剂聚二烯丙基二甲基氯化铵,制备石墨烯(GR)/聚二烯丙基二甲基氯化铵(PDDA)/铂纳米粒子(PtNPs)复合材料,在此基础上制备了GR/PDDA/PtNPs复合修饰电极,并采用透射电镜、电化学等方法对GR/PDDA/PtNPs进行表征。以pH=8.5的B.R缓冲溶液为支持电解质,采用循环伏安法研究了硝苯地平(Nifedipine,NF)和尼群地平(Nitrendipine,NT)在复合修饰电极上的电化学行为,并优化了电化学测量条件。NF溶液浓度为4.0×10(-7)~1.6×10(-4) mol·L^-1,NT溶液浓度为1.0×10^-6~1.4×10^-4 mol·L^-1,它们的还原峰电流与浓度成线性关系,检出限分别为5.0×10^-8 mol·L^-1,3.7×10^-7 mol·L^-1。所建立的方法成功应用于血药中NF、NT的测定,获得可靠满意的结果。     

碳纳米管/聚二烯丙基二甲基氯化铵/磷钼酸复合膜的组装及其在溴酸盐检测中的应用

通过静电层层组装技术在玻碳(GC)电极表面制备{多壁碳纳米管(MWCNT)/聚二烯丙基二甲基氯化铵(PDDA)}n多层膜,并采用循环伏安法在多层膜的表面电化学修饰一层磷钼酸(PMo12)膜,构筑GC/{MWCNT/PDDA}n-PMo12复合膜修饰电极.利用SEM对比观察{MWCNT/PDDA}n和{PDDA/MWCNT}n-PMo12的微观结构,并研究该复合膜修饰电极的电化学及其对溴酸盐(BrO3-)电催化还原性质.在此基础上研发一种基于GC/{MWCNT/PDDA}n-PMo12复合膜修饰电极的电流型BrO3-传感器,该传感器表现出明显增大的响应电流.在最优的实验条件下,采用电流时间曲线(i-t)法考察该复合膜修饰电极对BrO3-的安培响应.实验结果表明,该传感器在BrO3-浓度为50～400nmol/L的范围内具有良好的线性关系,相关系数R2为0.9950,响应时间为1.53s,检出限为20nmol/L,灵敏度为13.81mA(mmol/L)-1cm-2.