尿酸在聚6-甲基香豆素修饰的玻碳电极上的电化学行为

制备了聚6-甲基香豆素修饰玻碳电极,研究了尿酸(UA)在该修饰电极上的电化学行为。实验结果表明:在pH=5.0的磷酸盐缓冲溶液中,扫描速率为50mV/s时,尿酸在修饰电极上于0.352V处产生一个灵敏的氧化峰,在0.278V处有一弱的还原峰。经线性扫描伏安法测定,氧化峰电流与尿酸浓度在2.5×10-6～1.0×10-5mol/L范围内表现出良好的线性关系,检出限为1.0×10-6mol/L。将修饰电极在常温下放置50d及将体系温度升高到75℃时,修饰电极对尿酸的响应电流大体不变,结果满意。     

紫色非硫光合细菌Rhodopseudomonas capsulata铁氧还蛋白的分离纯化

采用超声破碎,Triton X-100处理,30％丙酮提取,经三次DEAE-52纤维素离子交换柱层析分离纯化,我们第一次从紫色非硫光合细菌Rps.capsulata N-3菌株中,获得聚丙烯酰胺凝胶电泳纯的铁氧还蛋白(Ferredoxin)及其结晶。吸收光谱的峰值位于275舳,375nm;在450 nm、480 nm处各有较小的吸收峰。特征吸收峰比A375nm/A275nm=0.74。凝胶过滤测定它的分子量为9,000道尔顿;每分子含有8个非血红素铁和等数量的酸性不稳定硫。铁氧还蛋白能被连二亚硫酸钠化学还原,氢气和氢酶构成的酶体系还原,亦能作为电子传递载体参与菠菜叶绿体催化的DCPIPH_2→铁氧还蛋白→NADP~ 光还原。     

黄曲霉毒素氧化酶/壳聚糖-单壁碳纳米管/聚邻苯二胺修饰电极对杂色曲霉素的检测

将黄曲霉毒素氧化酶(AFO)固定在壳聚糖(CS)-单壁碳纳米管(SWCNTs)杂交膜中,组装在聚邻苯二胺(POPD)修饰的金电极(Au)表面,制备了对杂色曲霉素(ST)敏感的生物传感器(AFO/CS-SWCNTs/POPD/Au)。运用原子力显微镜(AFM)、傅立叶变换红外光谱(FT-IR)和交流阻抗技术(EIS)对电极组装过程进行了表征。循环伏安法研究表明,AFO在修饰电极上发生了准可逆的氧化还原反应,是表面控制过程,其式量电位为-0.436V(vs.Ag/AgCl),说明包埋在CS-SWCNTs中的AFO和电极之间发生了直接电子传递。AFO修饰电极对ST具有明显的电催化作用,其表观米氏常数appKM为7.13μmol/L,催化电流与ST浓度在10～310ng/mL范围内呈线性关系,相关系数为0.997,检出限为3ng/mL(S/N=3),响应时间小于10s。组装的生物传感器具有较好的稳定性与重现性,连续检测20ng/mL的ST标准溶液11次,电流值RSD为3.9%;放置一个月后,其电流响应值仍为初始值的85.6%。该方法具有较高的选择性和灵敏度,应用于实际样品检测时,其回收率在87.6%～105.5%之间...     

葡萄糖氧化酶在枯草芽孢杆菌芽孢表面的展示及其酶电极制备

葡萄糖生物传感器是目前最为常见的电化学生物传感器,绝大多数葡萄糖生物传感器采用在电极表面修饰葡萄糖氧化酶的方法来制备,但是,在电极的固定化过程中需要酶的纯化,使得成本增加,已成为固定化酶电极开发领域的瓶颈。文中主要以芽孢衣壳蛋白CotX为锚定蛋白将葡萄糖氧化酶 (Glucose oxidase,GOD) 展示到枯草芽孢杆菌芽孢表面,通过Western blotting分析、免疫荧光分析以及酶活检测均证明GOD在芽孢表面有效表达,发酵获得重组芽孢 (Spore-GOD)。再采用滴涂法和电沉积法制备了氧化石墨烯/普鲁士蓝沉积膜修饰玻碳电极,将Spore-GOD固定在修饰电极表面,最后滴加一层Nafion溶液,制成了电化学生物传感器,用于葡萄糖的灵敏测定。葡萄糖在该酶电极传感器上的循环伏安图表明,该反应在0.42 V处出现明显的氧化峰,并且氧化还原峰电流与葡萄糖浓度在0.1–7.0 mmol/L之间具有良好的线性关系,校正曲线方程为：I=1.304 7Cglucose+3.639 (R2=0.992 9),其检测限为7.5 μmol/L (S/N=3)。此修饰电极具有良好的导电性能、稳定性和重现性,可用于葡萄糖的分析测定。     

纳米金-聚乙烯亚胺基因载体的制备

目的:基因方法治疗癌症近年来取得了很大的突破,因此基因载体的构建显得尤为重要.其中纳米基因载体合成简单,成本低廉,并能够包裹、浓缩、保护核苷酸使其免受核酸酶降解,因此纳米材料广泛地应用于基因输送.我们拟开展聚乙烯亚胺-纳米金基因载体的制备及其表征.方法:采用层层包裹技术制备基因载体,首先通过柠檬酸钠还原法制备纳米金颗粒后,应用11-巯基十一烷酸对金颗粒进行修饰,使其表面带有羧基,然后进一步将带有氨基的低分子量聚乙烯亚胺与羧基进行连接.应用动态光散射(DLS),紫外可见光谱(UV)和透射电子显微镜(TEM)对构建的纳米基因载体进行表征.结果:成功制备了聚乙烯亚胺-纳米金基因载体,检测表明每一步制备出的产物纳米尺寸在20-30nm之间,液体均匀稳定,分散系数(PDI)在0.2以下,Zeta电位测定表明,每步的产物电荷变化与外层包裹的反应物有关.尽管金颗粒外层包裹聚乙烯亚胺,但是总体上纳米载体尺寸没有发生太大的变化,TEM检测表明每一步形成了均匀的、单分散的、球状的纳米颗粒.结论:我们通过层层包裹技术成功制备了聚乙烯亚胺-纳米金基因载体,在进一步开展的生物活性的检测中,希望通过纳米载体的携带作用,将基因转染进靶细胞,从而检测相关基因对靶细胞的沉默作用,提高基因药物的应用,为开发新型基因药物提供基础.     

壳聚糖纳米磁性微球固定脂肪氧合酶的研究

实验采用溶胶凝胶法制备了纳米磁性Fe3O4,并用壳聚糖对颗粒2四川大学,生命科学学院,四川成都表面进行了表面修饰得到壳聚糖纳米磁性微球复合载体,再以戊二醛为交联剂将脂肪氧合酶固定在复合载体上,并测定了不同因素对游离酶和固定化酶活性的影响;实验表明,微粒在电镜观察下呈亮黑色球状,直径约为150nm,并具有良好的磁性,固定在载体上酶的含量约为7.6%,游离酶的最适温度为30℃,最适p H8.0,而固定化酶的最适温度为30℃,最适p H9.0,当H2O2浓度为12.0 g/L时,游离酶和固定化酶的活性最强;实验结果表明通过交联的方法成功将脂肪氧合酶固定在了纳米磁性四氧化三铁颗粒上,并表现出了较好的活性。     

耐高温重组CotA蛋白的胆红素氧化酶反应动力学及活性鉴定

已知源于枯草芽孢杆菌内生孢子的CotA蛋白具有漆酶和胆红素氧化酶活性。然而,其分离纯化极为困难。本研究对表达与纯化的重组CotA蛋白的胆红素氧化酶特性及氧化还原功能进行鉴定。基因转染及筛选获得了表达CotA的P. pastoris菌株|继而,表达的重组CotA蛋白经DEAE-Sepharose FF 及Sephadex G-75层析分离与纯化,产物得率为25%,纯化产物的酶比活性为 4 U/mg。经SDS-PAGE 和 MALDI-TOF MS 分析显示,其分子质量为65 kD。纯化的CotA蛋白能够催化胆红素氧化,生成胆绿素,且催化反应速率受反应溶液中溶解氧含量的影响,提示纯化的重组CotA具有胆红素氧化酶活性。酶反应进一步证明,CotA的胆红素氧化酶反应最适pH值为pH 8.0,最适温度为60℃。该酶在90℃条件下的半衰期为7 h,提示CotA胆红素氧化酶具有高度的热稳定性。CotA修饰的摄谱仪石墨电极可直接电催化分子氧（O2）还原,具有很好的电流响应。我们的结果表明,重组的CotA蛋白具有耐高温胆红素氧化酶活性。更重要的是,我们的结果还提示重组的CotA蛋白在酶生物燃料电池阴极的制备上具有较好的应用潜能。     

玉米过氧化物还原蛋白BAS1的原核表达及其功能研究

植物过氧化物还原蛋白BAS1是巯基依赖的过氧化物酶,通过催化的Cys残基还原过氧化氢,依赖NADPH的叶绿体硫氧还蛋白还原酶保持BAS1的还原态。玉米含有两种BAS1:2-Cys PrxA和2-Cys PrxB。利用RT-PCR方法从玉米幼叶中克隆了编码成熟2-Cys PrxA的基因,并将蛋白Cys34残基突变成Ser34。SDS-PAGE显示纯化的野生型和突变体蛋白为一条主带,分子量约为23kDa;体外蛋白结合实验表明纯化的叶绿体硫氧还蛋白还原酶通过分子间二硫键结合纯化的2Cys PrxA的C34S突变体,非还原SDS-PAGE显示纯化的野生型2Cys PrxA含有分子间二硫键组成的二体,而纯化的C34S突变体呈现单体,巯基专一性标记化合物AMS修饰及活性分析表明纯化的BAS1还原态是催化还原过氧化氢所所必须的,它由硫氧还蛋白还原酶及其辅酶NADPH所催化。     

微生物谷氨酰胺转氨酶催化细胞色素c赖氨酸残基的定点修饰

研究微生物谷氨酰胺转氨酶(mTG)催化细胞色素c(Cytc)的PEG定点修饰的可行性,并优化修饰条件,研究PEG修饰对Cytc性质的影响。将单甲氧基聚乙二醇氨(mPEG-NH_2)与N-苄氧羰基-谷氨酰胺-甘氨酸(CBZ-QG)共价结合制备含谷氨酰胺残基的甲氧基聚乙二醇衍生物(N-苄氧羰基-谷氨酰胺-甘氨酰-单甲氧基聚乙二醇,CBZ-QG-mPEG);mTG分别催化mPEG-NH_2、CBZQG-mPEG(mTG)修饰Cytc,研究酶法定点修饰Cytc残基的可行性;改变酶的用量、温度、反应时间和p H等反应条件优化谷胺酰胺转氨酶催化修饰Cytc的条件。研究结果表明:(1)mPEG-NH_2不能作为mTG的底物修饰Cytc,甲氧基聚乙二醇氨(mPEG-NH_2)分子上引入谷氨酰胺残基后,在mTG的催化作用下了实现Cytc的PEG修饰,而且基于mTG的底物特异性实现了PEG定点修饰Cytc的赖氨酸(Lys)残基;(2)37℃温度下,p H 8.0的溶液中,1mg/ml的mTG催化修饰反应2h是最佳修饰反应条件;(3)化学法PEG修饰Cytc产物复杂,是多种多点修饰产物的混合物,酶法催化PEG修饰Cytc只产生单一产物;(4)与天然Cytc相比,修饰后Cytc的活力、稳定性都有所提高。提出的谷胺酰胺转胺酶催化修饰法解决了蛋白质Lys残基难以定点修饰的难题,拓展了mTG在蛋白质修饰方面的应用。     

可见光响应的CdTe/TiO2复合纳米颗粒体外PDT灭活HL60细胞的研究

文章采用溶胶凝胶法制备核壳CdTe/TiO_2复合纳米颗粒,探讨了该复合纳米颗粒体外PDT对HL60细胞的灭活作用。通过扫描电镜(TEM)、X射线光电子衍射仪(XPS)对CdTe/TiO_2进行表征。文中,用紫外可见光吸收光谱(UV-vis)测得尺寸为2-5 nm的CdTe QDs吸收峰为460 nm。研究表明,CdTe/TiO_2复合纳米颗粒尺寸在80 nm左右,其吸收光谱相较于TiO_2的光响应区拓展至可见光区。将CdTe/TiO_2与HL60细胞进行共同孵育,采用CCK-8法研究了其在暗室条件下细胞的生长情况和浓度对细胞相对存活率的影响以及在不同浓度的CdTe/TiO_2复合纳米颗粒PDT后的细胞活性。实验结果表明:在共同孵育16 h后CdTe/TiO_2对HL60细胞的毒性最强,10~320μg/mL浓度的CdTe/TiO_2样品对HL60细胞均具有较强的灭活作用。当添加CdTe/TiO_2样品浓度为320μg/mL时,光照1 h后PDT灭活效率达到87.7%。     

Enhancement of DNA immobilization and hybridization on gold electrode modified by nanogold aggregates

Gold electrodes modified by nanogold aggregates (nanogold electrode) were obtained by the electrodeposition of gold nanoparticles onto planar gold electrode. The Electrochemical response of single-stranded DNA (ssDNA) probe immobilization and hybridization with target DNA was measured by cyclic voltammograms (CV) using methylene blue (MB) as an electroactive indicator. An improving method using long sequence target DNA, which greatly enhanced the response signal during hybridization, was studied. Nanogold electrodes could largely increase the immobilization amount of ssDNA probe. The hybridization amount of target DNA could be increased several times for the manifold nanogold electrodes. The detection limit of nanogold electrode for the complementary 16-mer oligonucleotide (target DNA1) and long sequence 55-mer oligonucleotide (target DNA2) could reach the concentration of 10(-9) mol/L and 10(-11) mol/L, respectively, which are far more sensitive than that of the planar electrode.     

A simple and sensitive resonance Rayleigh scattering method for determination of As(III) using aptamer‐modified nanogold as a probe

A simple and selective aptamer (ssDNA)‐modified nanogold probe (AussDNA) was prepared for the determination of trace As(III) in HEPES buffer solution (pH 8.2) containing 0.05 mol/L NaCl. The method coupled the aptamer reaction of AussDNA–As(III) and the resonance Rayleigh scattering (RRS) of nanogold aggregations at 278 nm. When the As(III) concentration increased, the RRS intensity at 278 nm increased to form more nanogold aggregation and a stable As(III)–ssDNA complex. Under selected conditions, the increased RRS intensity (ΔI) was linear to the concentration of As(III) in the range 3.8–230.4 ng/mL, with a detection limit of 1.9 ng/mL. This RRS method was applied to detect As(III) in water samples, with simplicity, sensitivity and selectivity. Copyright © 2013 John Wiley & Sons, Ltd.     

Electrochemical catalysis and thermal stability characterization of laccase-carbon nanotubes-ionic liquid nanocomposite modified graphite electrode

The hydrophobic carbon nanotubes-ionic liquid (CNTs-IL) gel forms a stable modified film on hydrophobic graphite electrode surface. Laccase immobilized on the CNTs-IL gel film modified electrode shows good thermal stability and enhanced electrochemical catalytic ability. The optimal bioactivity occurs with increasing temperature and this optimum is 20 degrees C higher in comparison to free laccase. The improvement of laccase thermal stability may be due to the microenvironment of hydrophobic CNTs-IL gel on graphite electrode surface. On the other hand, the sensitive detection of oxygen has been achieved due to the feasibility of oxygen reduction by both of laccase and nanocomposite of CNTs-IL gel. Furthermore, the laccase hybrid nanocomposite also shows the fast electrochemical response and high sensitivity to the inhibitors of halide ions with the approximate IC50 of 0.01, 4.2 and 87.5 mM for the fluoride, chloride and bromide ions, respectively. It implies the feasibility of laccase modified electrode as an inhibition biosensor to detect the modulators of laccase.     

Laccase electrode for direct electrocatalytic reduction of O2 to H2O with high-operational stability and resistance to chloride inhibition

Laccase from Trametes hirsuta basidiomycete has been covalently bound to graphite electrodes electrochemically modified with phenyl derivatives as a way to attach the enzyme molecules with an adequate orientation for direct electron transfer (DET). Current densities up to 0.5mA/cm(2) of electrocatalytic reduction of O(2) to H(2)O were obtained in absence of redox mediators, suggesting preferential orientation of the T1 Cu centre of the laccase towards the electrode. The covalent attachment of the laccase molecules to the functionalized electrodes permitted remarkable operational stability. Moreover, O(2) bioelectroreduction based on DET between the laccase and the electrode was not inhibited by chloride ions, whereas mediated bioelectrocatalysis was. In contrast, fluoride ions inhibited both direct and mediated electron transfers-based bioelectrocatalytic reduction of O(2). Thus, two different modes of laccase inhibition by halides are discussed.     

A biofuel cell with enhanced performance by multilayer biocatalyst immobilized on highly ordered macroporous electrode

A one-compartment glucose/O(2) biofuel cell based on an electrostatic layer-by-layer (LbL) technique on three-dimensional ordered macroporous (3DOM) gold electrode was described. A 3DOM gold electrode was synthesized electrochemically by an inverted colloidal crystal template technique. Then the macroporous gold electrodes were functionalized with Au nanoparticles (AuNPs) and enzyme, glucose dehydrogenase (GDH) or laccase. The (AuNPs/GDH)(n) multilayer modified macroporous gold electrode showed excellent bioelectrocatalytic activity towards glucose. The direct electroreduction towards oxygen was realized at (AuNPs/laccase)(n) films on 3DOM gold electrodes. The maximum power density of the cell with the macroporous film as matrix was 178muWcm(-2) at 226mV, which was 16 times larger than that of the biofuel cell with the flat electrode under the same condition. The proposed method is simple and would be applicable to enhance the power output of miniaturized biofuel cell.     

Real-time investigation of the interaction between primaquine phosphate and bovine serum albumin (BSA) by piezoelectric quartz crystal impedance analysis

Real-time investigation of the interaction between primaquine phosphate and bovine serum albumin by the piezoelectric quartz crystal impedance (PQCI) analysis was carried out for the first time. Three kinds of electrodes were investigated. Compared with bare gold (Au) electrode, the gold electrode self-assembled of nanogold colloids exhibits maintained biocompatibility, increased capacity and more bioactivity. Additionally, on the basis of the multi-dimensional information provided by the PQCI analysis, the real-time interaction information and the kinetics of the binding process was investigated and a response model was deduced. At 37 degrees C, the binding rate (k1), dissociation rate (k(-1)) and equilibrium constants (Ka) were 4.19x10(2) (mol l(-1))(-1) s(-1), 1.01x10(-3) s(-1) and 4.15x10(5) (mol l(-1))(-1) for the electrode modified by nanogold particles; 3.83x10(2) (mol l(-1))(-1) s(-1), 9.70x10(-4) s(-1) and 3.95x10(5) (mol l(-1))(-1) for the bare gold electrode, respectively.     

A hydroquinone biosensor using modified core-shell magnetic nanoparticles supported on carbon paste electrode

A hydroquinone biosensor was developed and used to determine hydroquinone concentration in compost extracts based on the immobilization of laccase on the surface of modified magnetic core-shell (Fe(3)O(4)-SiO2) nanoparticles. Laccase was covalently immobilized on the magnetic nanoparticles by glutaraldehyde, which was modified with amino groups on its surface. The obtained magnetic bio-nanoparticles were attached to the surface of carbon paste electrode with the aid of a permanent magnet to determine hydroquinone. A good microenvironment for retaining the bioactivity of laccase was provided by the immobilization matrix. The linear range for hydroquinone determination was 1 x 10(-7) to 1.375 x 10(-4)M, with a detection limit of 1.5 x 10(-8)M. The current reached 95% of the steady-state current within about 60s. Hydroquinone concentration in compost extracts was determined by laccase biosensor and HPLC, the results of the two methods were approximately the same.     

A Simple and Rapid Resonance Scattering Spectral Method for Detection of Trace Hg<Superscript>2+</Superscript> Using Aptamer-Nanogold as Probe

Single-strand deoxyribonucleic acid (ssDNA) were used to modified nanogold particle to obtain a aptamer-nanogold probe (NGssDNA) for Hg(II). The probe is not aggregated in high concentration of NaCl. In the pH 7.0 Na₂HPO₄-NaH₂PO₄ buffer solution and in the presence of high concentration of NaCl, NGssDNA interact with Hg(II) to form stable double-strand T-Hg(II)-T mismatches and to release nanogold particles from the probe. The released nanogold particles aggregated to form bigger clusters which leaded the resonance scattering (RS) intensity at 540 nm enhanced linearly with the concentration of Hg²⁺ in the range of 0.39–1666.7 nM, with detection of 0.1 nM. This simple, rapid, and sensitive aptamer-nanogold RS assay was applied to determination of Hg²⁺ in wastewater, with satisfactory results.     

Fluorescein-labeled “arch-like” DNA probes for electrochemical detection of DNA on gold nanoparticle-modified gold electrodes

In the present study, a gold nanoparticle-modified gold electrode (nanogold electrode) was used to develop a novel fluorescein electrochemical DNA biosensor based on a target-induced conformational change. The nanogold electrode was obtained by electrodepositing gold nanoparticles onto a bare gold electrode. This modification not only immobilized probe oligonucleotides, but also adsorbed fluorescein onto the surface of the gold nanoparticles to form an “arch-like” structure. This article compares the electrochemical signal changes caused by the hybridization of “arch-like” DNA on nanogold electrode and linear DNA on bare gold electrode. The results showed that the adsorption effect of nanogold can enhance the sensitivity of the sensor. The linear range of target ssDNA is from 2.0 × 10⁻⁹ M to 2.0 × 10⁻⁸ M with a correlation coefficient of 0.9956 and detection limit (3σ) of 7.10 × 10⁻¹⁰ M. Additionally, the specificity and hybridization response of this simple sensor were investigated.     

A Highly Sensitive Resonance Scattering Assay for Immunoglobulin M Using Ag(I)–Hydroquinone–Immunonanogold Catalytic Reaction

Ten-nanometer nanogold showed the strongest catalytic effect on the particle reaction between Ag(I) and hydroquinone to form nanosilver particles that exhibited the strongest resonance scattering (RS) peak at 350 nm. The enhanced RS intensity was linear to the nanogold concentration in the range of 30–5,700 nM Au. The nanogold was used to label goat antihuman immunoglobulin M (GIgM) to obtain an immunonanogold probe (AuGIgM) for immunoglobulin M (IgM). Based on the nanogold-labeled immunoreaction between IgM and AuGIgM, centrifugation, and AuGIgM–Ag(I)–hydroquinone nanocatalytic reaction, a highly sensitive and selective immunonanogold-catalytic Ag particle RS assay for 0.2–300 ng mL^?1 IgM was proposed, with a detection limit of 0.1 ng mL^?1. This assay was simple and sensitive and was applied to assay IgM in serum samples, with satisfactory results.