氧化硅介孔泡沫材料固定木瓜蛋白酶的研究

以氧化硅介孔泡沫材料(Siliceous Mesocellular Foam, MCF)为载体对木瓜蛋白酶进行了固定, 并对固定化酶的性能和影响因素以及酶的稳定性作了系统的研究. 结果表明, 木瓜蛋白酶在MCF上能获得较大的负载量（334mg/g MCF）. 固定化木瓜蛋白酶的最适反应温度较游离酶提高了10℃, 最适pH向碱性方向偏移0.5个单位. 固定化木瓜蛋白酶的米氏常数（Km）为6.99×10^-3mol/L, 在八批次操作后酶活保留65.1%. 在4℃条件下放置60d后, 固定化酶的剩余活性仍保持75%以上, 而游离酶的活性只有初始活性的53.6%. 与游离酶相比, 固定化木瓜蛋白酶的pH稳定性、热稳定性、操作稳定性和储藏稳定性都有明显改善, 有利于酶的重复使用和储藏. 介孔泡沫材料是一种良好的木瓜蛋白酶固定载体.     

基于短孔道Zr-Ce-SBA-15固定胃蛋白酶的活性生物催化剂

在不外加无机酸的条件下, 通过水热合成法制备了短孔道六方板状有序介孔材料Zr-Ce-SBA-15 (ZCS). 以ZCS和传统SBA-15为载体对胃蛋白酶进行固定, 并利用N-(2-氨乙基)-3-氨丙基三甲氧基硅烷(AAPTS)对酶固定化材料进行功能化, 以缩小开口孔径从而减少酶泄漏. 采用小角X射线衍射、扫描电镜、透射电镜、氮气吸附和红外光谱对样品进行结构表征. 结果表明, 胃蛋白酶成功固定到介孔孔道中, AAPTS嫁接到材料中且没有破坏介孔结构. 固定化实验表明相比于SBA-15, ZCS对胃蛋白酶具有较快的吸附速度和较强的固定化能力(最大负载量为257.9 mg/g), 短孔道材料能有效地促进分子的扩散传递. 催化活性测定以牛血红蛋白为探针物, 与游离酶相比, 固定酶对牛血红蛋白保持着稳定的活性.     

基于layer-by-layer技术构建胆碱检测生物传感器酶电极的研究

基于层层累积自组装法将PDDA高分子材料和胆碱氧化酶逐层固定在高分子聚合膜PVS/PDDA修饰的电极表面,制备了电流型胆碱检测生物传感器.利用石英晶体微天平(QCM)分别分析了PDDA和胆碱氧化酶的固定过程,结果表明酶的固定量可以得到有效控制.探讨了自组装膜层数、pH值、温度对传感器电流响应的影响.制备的生物传感器在胆碱浓度为5×10-7～1×10-4 mol/L的范围内对胆碱有良好的线性响应,响应时间为10 s,检出限为5×10-7 mol/L.传感器的稳定性好,30天时的响应值仍保持90%.     

环氧基功能化块体SiO_2大孔材料对漆酶的固定化

采用后嫁接法在溶剂热条件下对新型块体SiO2大孔材料进行环氧基化改性,以环氧基功能化SiO2大孔材料作为载体,通过共价结合法固定化诺维信(Novozymes)工业级漆酶,对固定化条件进行了优化,研究了固定化酶与游离酶的酶学性质。结果表明,在固定化时间为4 h、pH值为4.5、初始酶液浓度为25 mg/mL时,固定化效果最好,固定化漆酶活力达到101.7 U/g;固定化漆酶的最适pH值为4.0,最适温度为50℃,其pH值稳定性和热稳定性都显著优于游离漆酶。固定化漆酶具有可重复操作的性质,与底物反应反复操作10批次后剩余活性为43.4%。     

同时具有光敏感性和酶催化功能的复合敏感膜

采用溶液浇铸法成膜,把荧光指示剂和葡萄糖氧化物酶(GOD)同时固定于醋酸纤维素膜上,得到同时具有光敏感性和酶催化能力的复合敏感膜.利用SEM和紫外-可见光分光光度计对复合敏感膜表面形貌和酶活性进行了分析.荧光指示剂没有泄漏,固定化酶的稳定性高于游离酶,表明此醋酸纤维素膜可作为一种优良的荧光指示剂和酶固定化载体.     

SiO2/海藻酸钠复合水凝胶作为固定化纤维素酶载体

以海藻酸钠和γ-氨丙基三乙氧基硅烷为原料,在一定条件下使二者发生交联反应生成复合水凝胶,并以此复合凝胶作为固定化纤维素酶的载体。纤维素酶的包埋率超过了85%, 酶固定前后的SEM图表明纤维素酶非常均匀地分布在载体中。探讨了pH、 温度对固定化酶和游离酶活力的影响, 结果表明固定化酶具有对pH和温度更高的稳定性, 固定化酶的最适宜pH为3.6,最佳催化温度为50℃。通过Michaelis Menten（米氏）方程计算得到的固定化酶的米式常数（K_m）值较游离酶大,表明固定化酶的可重复使用性和储藏稳定性良好,连续使用10次后依然保留>50%酶活力,储藏1个月后固定化酶依然有81%的相对酶活力。      

碳酸酐酶在硅藻土上的固定化及其性质研究

以硅藻土为载体,通过共价结合法固定化碳酸酐酶.对制备固定化晦过程中的pH值、加酶量、反应温度、反应时间几个重要的影响因素进行了研究和优化,并对固定化酶和游离酶的酶学性质进行了比较.结果表明,固定化酶的最适反应温度为30℃,比游离酶高了5℃;最适反应pH值为6.0,比游离酶低了2个pH值单位.该固定化酶的热稳定性、酸碱稳定性和操作稳定性均高于游离酶.     

基于Pt-Pb纳米花的无酶葡萄糖传感器性能研究

利用超声恒电流沉积法在金电极表面制备了Pt-Pb纳米花,并将其用于构建无酶葡萄糖传感器,采用阻抗谱、循环伏安和差分脉冲伏安法研究了其电化学性能。SEM结果表明,当沉积电流为10mA时,电极表面形成Pt-Pb纳米花结构。电化学测试结果表明,该电极对葡萄糖具有很好的电催化性能,在含有氯离子的溶液中不会失活。Pt-Pb纳米花电极构建的无酶传感器对葡萄糖的线性响应范围为0.5×10-3～22×10-3 mol/L,灵敏度为3.68mA·cm-2·(mol/L)-1,检测限为24×10-6 mol/L。此外,传感器具有良好的选择性、重复性和稳定性。     

La_(0.7)Sr_(0.3)NiO_3/壳聚糖/聚吡咯/牛血红蛋白修饰玻碳电极的制备及构建过氧化氢传感器

在玻碳电极上修饰一层表面均匀的聚吡咯膜,然后利用钙钛矿和壳聚糖的复合膜来固定牛血红蛋白(Hb),制备出性能良好的过氧化氢生物传感器.采用循环伏安(CV)和扫描电镜对修饰电极进行了表征.该传感器对H2O2具有好的催化响应,且响应快.在优化的实验条件下,所制备的传感器对H2O2的线性范围为7.0×10-6～1.5×10-3mol/L,检测限为9.0×10-8mol/L.     

大孔/介孔多级孔SiO2的制备及其在固定化脂肪酶中的应用

以具有三维骨架结构的环氧树脂大孔聚合物为模板,制备具有毫米级尺寸的大孔/介孔多级孔SiO_2。应用SEM、MIP、FTIR和N_2吸附-脱附法对材料孔道结构和表面性质进行表征。采用吸附法固定褶皱假丝酵母脂肪酶(CRL),研究CRL初始浓度、pH值及固定化时间对脂肪酶固定化的影响,对比研究了游离脂肪酶和固定化脂肪酶的酶学性质。结果表明,大孔/介孔SiO_2具有三维连续贯通的大孔孔道,孔壁由连续的SiO_2纳米薄膜构筑而成且表面存在丰富的介孔,比表面积为75.1m~2/g,孔隙率为92.3%;在CRL浓度为0.6mg/mL、pH值为8.0、固定化时间为10h时,固定化酶酶活达到4 825U/g。与游离脂肪酶相比,固定化脂肪酶的pH稳定性、热稳定性和储存稳定性明显提高,连续使用8次后的酶活为初始酶活的68%。利用环氧树脂大孔聚合物模板制备的大孔/介孔多级孔SiO_2在固定化酶方面具有良好的应用前景。     

Significance of antibody orientation unraveled: well-oriented antibodies recorded high binding affinity

To investigate the effect of antibody orientation on its immunological activities, we developed a novel and versatile platform consisting of a well-defined phospholipid polymer surface on which staphylococcal protein A (SpA) was site-selectively immobilized. The application of a biocompatible phospholipid-based platform ensured minimal denaturation of immobilized antibodies, and the site-selective immobilization of SpA clarified the effect of antibody orientation on immunological activities. The phospholipid polymer platform was prepared on silicon substrates using the surface-initiated atom transfer radical polymerization (SI-ATRP) technique. An enzymatic reaction was performed for orientation-selective coupling of SpA molecules to the polymer brush surface. Orientation-controlled antibodies were achieved using enzymatic reactions, and these antibodies captured 1.8 ± 0.1 antigens on average, implying that at least 80% of immobilized antibodies reacted with two antigens. Theoretical multivalent binding analysis further revealed that orientation-controlled antibodies had antigen-antibody reaction equilibrium dissociation constants (K(d)) as low as 8.6 × 10(-10) mol/L, whereas randomly oriented and partially oriented antibodies showed K(d) values of 2.0 × 10(-7) and 1.2 × 10(-7) mol/L, respectively. Strict control of antibody orientation not only formed an approximately 100-fold stronger antigen-antibody complex than the controls but also sustained the native antibody K(d) (10(-10)-10(-9) mol/L). These findings support the significance of antibody orientation because controlling the orientation resulted in high reactivity and theoretical binding capacity.     

Dithiobissuccinimidyl propionate as an anchor for assembling peroxidases at electrodes surfaces and its application in a H2O2 biosensor

Exposure of gold surfaces to solutions of dithiobis N-succinimidyl propionate (DTSP) gives rise to the modification of the surface with N-succinimidyl-3-thiopropionate (NSTP) which can, in turn, react with amino groups allowing for the covalent immobilization of enzymes such as horseradish peroxidase (HRP). The coverage of NSTP has been estimated to be of the order of 1.3 x 10(-10) from the charge consumed during its reductive desorption. The binding reaction of HRP with NSTP modified gold surfaces has been studied with the quartz crystal microbalance, and the results suggest that the immobilization process involves two steps in which the first (faster) appears to correspond to the rapid incorporation of the enzyme whereas the second is likely due to the slow incorporation of additional enzyme and/or reorganization of the immobilized layer. Spectrophotometric and electrochemical assays indicate that the immobilized HRP retains its enzymatic activity after immobilization onto the DTSP modified gold surface. The amount of immobilized (and active) HRP was estimated from QCM and spectrophotometric measurements to be of the order of 1.5 x 10(-11) mol/cm2. A peroxide biosensor was developed making use of a gold surface modified with DTSP and HRP employing Os and Ru complexes of 1,10-phenanthroline 5,6-dione (phen-dione) of the type [M(phendione)x(L)3-x]+2 (where L = 1,10-phenanthroline or 2,2'-bipyridine, x = 1-3) as mediators with the quinone moieties being the active component. The efficiency of the mediators increased with increasing number of phendione ligands.     

Room temperature ionic liquid (RTIL)-decorated mesoporous silica SBA-15 for papain immobilization: RTIL increased the amount and activity of immobilized enzyme

Mesoporous materials, especially functionalized ones, have become a promising carrier for enzyme immobilization. We synthesized room temperature ionic liquid-decorated mesoporous SBA-15 (RTIL-SBA-15) for papain immobilization. The results of powder XRD, IR and N₂ adsorption-desorption isotherms have confirmed that ionic liquid [Simim⁺][Cl⁻] was successfully grafted on the surface of SBA-15. As a consequence of the electrostatic attraction between the cation [Simim⁺] and the negatively charged papain, RTIL-SBA-15 had an advantage over SBA-15 when papain was immobilized at pH = 9.00. The kinetic study showed that the interaction between papain and the carrier was stronger after ionic liquid modification. In the casein hydrolysis, the papain immobilized on RTIL-SBA-15 showed a higher specific activity than that on SBA-15, implying that the ionic liquid [Simim⁺][Cl⁻] was beneficial to improve the activity of the immobilized papain. The optimum pH of the immobilized papain was shifted to higher than that of free enzyme.     

Preparation and characterization of monodispersed microfloccules of TiO₂ nanoparticles with immobilized multienzymes

Microfloccules of TiO(2) nanoparticles, on which glycerol-dehydrogenase (GDH), 1,3-propanediol-oxidoreductase (PDOR), and glycerol-dehydratase (GDHt) were coimmobilized, were prepared by adsorption-flocculation with polyacrylamide (PAM). The catalytic activity of immobilized enzyme in the glycerol redox reaction system, the enzyme leakage, stabilities of pH and temperature, as well as catalytic kinetics of immobilized enzymes relative to the free enzymes were evaluated. Enzyme loading on the microfloccules as much as 104.1 mg/g TiO(2) (>90% loading efficiency) was obtained under the optimal conditions. PAM played a key role for the formation of microfloccules with relatively homogeneous distribution of size and reducing the enzyme leakage from the microfloccules during the catalysis reaction. The stabilities of GDH against pH and temperature was significantly higher than that those of free GDH. Kinetic study demonstrated that simultaneous NAD(H) regeneration was feasible in glycerol redox system catalysted by these multienzyme microfloccules and the yield of 1, 3-popanediol (1, 3-PD) was up to 11.62 g/L. These results indicated that the porous and easy-separation microfloccules of TiO(2) nanoparticles with immobilized multienzymes were efficient in term of catalytic activity as much as the free enzymes. Moreover, compared with free enzyme, the immobilized multienzymes system exhibited the broader pH, higher temperature stability.     

A new Schiff's base ligand immobilized agarose membrane optical sensor for selective monitoring of mercury ion

A highly selective optical sensor was developed for the Hg(2+) determination by chemical immobilization of 2-[(2-sulfanylphenyl)ethanimidoyl]phenol (L), on an agarose membrane. Spectrophotometric studies of complex formation between the Schiff's base ligand L and Hg(2+), Sr(2+), Mn(2+), Cu(2+), Al(3+), Cd(2+), Zn(2+), Co(2+) and Ag(+) metal ions in methanol solution indicated a substantially larger stability constant for the mercury ion complex. Consequently, the Schiff's base L was used as an appropriate ionophore for the preparation of a selective Hg(2+) optical sensor, by its immobilization on a transparent agarose film. A distinct color change, from yellow to green-blue, was observed by contacting the sensing membrane with Hg(2+) ions at pH 4.5. The effects of pH, ionophore concentration, ionic strength and reaction time on the immobilization of L were studied. A linear relationship was observed between the membrane absorbance at 650 nm and Hg(2+) concentrations in a range from 1×10(-2) to 1×10(-5) mol L(-1) with a detection limit (3σ) of 1×10(-6) mol L(-1). No significant interference from 100 times concentrations of a number of potentially interfering ions was detected for the mercury ion determination. The optical sensor was successfully applied to the determination of mercury in amalgam alloy and spiked water samples.     

Application of polymer quantum dot-enzyme hybrids in the biosensor development and test paper fabrication

Both glutathione capped CdTe quantum dots (QDs) and enzymes were encapsulated with poly(diallyldimethylammonium chloride) (PDDA) via electrostatic attraction to form hybrid films. The obtained PDDA QD-enzyme hybrids feature both high fluorescence and biorecognition. In the obtained hybrid materials, the fluorescence emission of the QDs was stable for at least 3 months, and the structure and activity of the enzyme was also well maintained as the Michaelis constant of tyrosinase was determined to be 0.90 mmol/L, which is just 2 times higher than that of free enzyme. This hybrid material was then utilized as a platform for the development of biosensors based on the quenching effects of the enzymatic products on the emission of the QDs with a kind of phenol (catechol) and glucose as example analytes. The detection limits of catechol and glucose were 1.0 × 10(-5) and 5.0 × 10(-6) mol/L, respectively. Moreover, this hybrid material was applied to the fabrication of test paper for these two analytes. The test paper was very stable with respect to the fluorescence of the QDs and the activity of the enzyme maintained for at least 1 month.     

Preparation and analytical application of mimetic enzyme hemin-loading magnetic silica nanotubes

We report here the preparation and analytical application of mimetic enzyme-loading magnetic silica nanotubes (MSNs), which were synthesized via alumina template membrane method. The nanotubes integrate the advantages of silica nanotubes and superparamagnetic characteristics. Hemin (a peroxidase mimic) was selected as a model for enzyme assays to demonstrate the applicability of these MSNs in enzyme immobilization. The immobilized hemin exhibited excellent catalytic activity and reusability. Using the immobilized hemin as the catalyst, the fluorometric measurement of thiamine has been achieved with a detection limit of 2.5 x 10(-8) mol/L. Our work herein reveals that the surface functionalized MSNs will be a promising platform as biocatalyst carriers.     

纳米铜粉的制备及其分散性研究

采用液相还原法制备纳米铜粉和纳米铜胶体,选用十六烷基三甲基溴化铵(CTAB)或聚乙烯吡咯烷酮(PVP)为分散剂,通过X射线衍射仪、扫描电镜对纳米铜粉进行表征,通过重力沉降法测试纳米铜胶体的稳定性。结果表明:液相还原法能够制备纯度较高的纳米铜粉;CTAB浓度为0.12 mol/L时,铜粉平均粒径最小,为20 nm,CTAB浓度为0.14 mol/L且超声粉碎20 min时,纳米铜胶体最稳定;PVP浓度为6×10^-4 mol/L时,铜粉平均粒径最小,为20 nm,PVP浓度为7×10^-4 mol/L且超声粉碎20 min时,纳米铜胶体最稳定。