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。     

Functionalized Multi‐Wall Carbon Nanotubes for Lipase Immobilization

We examine the immobilization of lipase B from Candida antarctica on functionalized multi‐wall carbon nanotubes (MWCNTs) through physical adsorption. MWCNTs functionalized with carboxyl‐, amine‐ and ester‐ terminal groups on their surface are used as immobilization carriers. Dispersion of the nanotubes and the immobilization procedure take place in aqueous and low‐water media. High enzyme loadings are attained, up to 25% of the weight of the carbon nanotubes. These novel biomaterials are characterized though FT‐IR and Raman spectroscopy. The MWCNT–lipase bioconjugates exhibit high catalytic activity and increased storage and operational stability. The biomaterials retain more than 55% of their initial activity after 6 months at 4 °C, while they retain approximately 25% of their initial activity after 30 d of incubation in hexane at 60 °C. The catalytic behaviour of the immobilized enzyme depends on the terminal group of the carbon nanotubes, the concentration of the enzyme and the immobilization method employed.     

Optimal environment for glucose oxidase in perfluorosulfonated ionomer membranes: improvement of first-generation biosensors

An optimal environment for glucose oxidase (GOx) in Nafion membranes is achieved using an advanced immobilization protocol based on a nonaqueous immobilization route. Exposure of glucose oxidase to water-organic mixtures with a high (85-95%) content of the organic solvent resulted in stabilization of the enzyme by a membrane-forming polyelectrolyte. Such an optimal environment leads to the highest enzyme specific activity in the resulting membrane, as desired for optimal use of the expensive oxidases. Casting solution containing glucose oxidase and Nafion is completely stable over 5 days in a refrigerator, providing almost absolute reproducibility of GOx-Nafion membranes. A glucose biosensor was prepared by casting the GOx-Nafion membranes over Prussian Blue-modified glassy carbon disk electrodes. The biosensor operated in the FIA mode allows the detection of glucose down to the 0.1 microM level, along with high sensitivity (0.05 A M(-1) cm(-2)), which is only 10 times lower than the sensitivity of the hydrogen peroxide transducer used. A comparison with the recently reported enzyme electrodes based on similar H2O2 transducers (transition metal hexacyanoferrates) shows that the proposed approach displays a dramatic (100-fold) improvement in sensitivity of the resulting biosensor. Combined with the attractive performance of a Prussian Blue-based hydrogen peroxide transducer, the proposed immobilization protocol provides a superior performance for first-generation glucose biosensors in term of sensitivity and detection limits.     

The immobilization of trypsin on soap-free P(MMA-EA-AA) latex particles

Poly(methyl methacrylate-ethyl acrylate-acrylic acid) latex particles with narrow size distribution and with surface carboxyl groups were produced by soap-free emulsion polymerization, and covalent immobilization of trypsin onto these particles was carried out by using the water-soluble carbodiimide (EDC) as an activating agent under various conditions. Different immobilization methods were employed and the factors affecting the efficiency and activity of the immobilized enzyme, such as the amount of trypsin and EDC, pH and temperature of the immobilization reaction were investigated. Results showed that both relatively high immobilization efficiency and high enzyme activity were achieved when pre-adsorption method was employed. The immobilization efficiency decreased as the trypsin amount increased, and increased as pH and temperature increased. When the EDC amount varied, the immobilization efficiency first increased significantly and then decreased slowly. A maximum of enzyme activity can be obtained at the optimum value of 958.0 mg trypsin/g dried particles and 372.5 mg EDC/g dried particles at 25 °C and pH 5.0. The immobilized trypsin exhibited much higher relative activity than its free counterpart.     

Simple synthesis of hierarchically ordered mesocellular mesoporous silica materials hosting crosslinked enzyme aggregates

Hierarchically ordered mesocellular mesoporous silica materials (HMMS) were synthesized using a single structure-directing agent. The mesocellular pores are synthesized without adding any pore expander; the pore walls are composed of SBA-15 type mesopores. Small-angle X-ray scattering revealed the presence of uniform pore structures with two different sizes. Using HMMS as a nanoscopic template, hierarchically ordered mesocellular mesoporous carbon (HMMC) and polymer (HMMP) materials were synthesized. HMMS was used as a host for enzyme immobilization. To improve the retention of enzymes in HMMS, we adsorbed enzymes, and then employed crosslinking using glutaraldehyde (GA). The resulting crosslinked enzyme aggregates (CLEAs) show an impressive stability with extremely high enzyme loadings. For example, 0.5 g alpha-chymotrypsin (CT) could be loaded in 1 g of silica with no activity decrease observed with rigorous shaking over one month. In contrast, adsorbed CT without GA treatment resulted in a lower loading, which further decreased due to continuous leaching of adsorbed CT under shaking. The activity of crosslinked CT aggregates in HMMS was approximately 10 times higher than that of the adsorbed CT, which represents a 74-fold increase in activity per unit weight of HMMS due to higher CT loading.     

聚丙烯接枝聚乙二醇改性聚丙烯微孔膜

以聚丙烯接枝聚乙二醇(PP-g-PEG)对聚丙烯共混改性,通过单向拉伸工艺制备亲水性聚丙烯微孔膜。使用压汞仪、扫描电镜、Gurley值和水蒸气透过率表征微孔膜的孔结构和透过性能,研究PP-g-PEG含量、PEG接枝率及链长对微孔膜孔结构、水蒸汽透过率及锂电池性能的影响。结果表明,对于接枝率为0.78%的PP-g-PEG,随着添加剂PP-g-PEG含量增加,微孔膜孔隙率和微孔尺寸下降,反映孔道结构的曲挠度及喉孔比上升;微孔膜孔结构与亲水性的综合作用导致水蒸气透过率在PP-g-PEG含量为2.5%时达到最大值。PP-g-PEG含量为2.5%和5.0%的微孔膜组装的锂电池具有较低的电荷转移电阻和较高的充放电比容量,电池性能优于未改性微孔膜组装的电池。提高PEG接枝率使得改性微孔膜孔结构有所变差,但是水蒸气透过率提高,组装电池的性能也更好。长PEG侧链PP-g-PEG对微孔膜孔结构的破坏程度大于短PEG侧链PP-g-PEG,导致组装电池的性能变差。     

Cage like ordered carboxylic acid functionalized mesoporous silica with enlarged pores for enzyme adsorption

Cubic FDU-12 type mesoporous silicas with enlarged pores and carboxylic acid (–COOH) functionality in the pore channels (denoted as LP-FTC-x) are synthesized using tetraethyl orthosilicate (TEOS) and carboxyethylsilanetriol sodium salt (CES) as silica sources, Pluronic F127 triblock copolymer as template, and trimethylbenzene (TMB) as pore expander, and utilized them as supports for enzyme immobilization. When the –COOH content is increased from 0 to 30%, the pore size of LP-FTC-x decreases from 23.6 to 11.1 nm, and its particle size decreases from around 2 μm to 600–800 nm. The material exhibits a high papain adsorption capacity (895 mg g^?1) with a low leaching rate at pH 8.2 due to the well-defined surface chemistry in the pore channel. The adsorption kinetics and isotherms follow the pseudo-second-order model and the Langmuir isotherm model, respectively. The excellent structural properties of LP-FTC-x are also advantageous for enhancement in stability of enzyme toward the temperature, solution pH, and incubation time variations.     

Octadecylsilylated porous silica rods as separation media for reversed-phase liquid chromatography

Continuous porous silica rods consisting of a mesoporous (pore size, 14 or 25 nm) silica skeleton of ～1 μm size and through-pores of ～1.7 μm were prepared and derivatized to C(18) phase by on-column reaction with octadecyldimethyl-(N,N-diethylamino)silane. The C(18) silica rods gave plate heights of 10-20 μm for aromatic hydrocarbons in 80% methanol and 20-40 μm for insulin in acetonitrile-water mixtures in the presence of trifluoroacetic acid. The performance of the silica rods was much better at a high flow rate than that of conventional columns packed with 5 μm C(18) silica particles having 12 and 30 nm pores, especially for high molecular weight species.     

On-chip encapsulation of lipase using mesoporous silica: A new route to enzyme microreactors

A polydimethylsiloxane/glass microfluidic reactor containing lipase-mesoporous silica (FSM-22) conjugates has been successfully constructed without chemical cross-linking between enzyme and support. A direct visualization of conjugates of lipase and FSM-22 immobilized in the microreactor by optical microscopy revealed that the enzymes were uniformly dispersed throughout the particles of the FSM-22, because of the successful immobilization of the enzyme. Moreover, the lipase-FSM-22 conjugates contained in the microreactor indicated higher enzymatic activity in hydrolysis of triglyceride, as compared with a batch experiment. These results demonstrate that the microreactor using mesoporous silica performs not only the reagent-less enzyme immobilization but also the high reactivity of the enzyme.     

Biocatalytic materials having a porous structure

Biocatalytic materials having a porous structure have been prepared by radiation polymerization at low temperatures using hydrophilic 2-hydroxyethyl methacrylate monomer and-glucosidase. The biocatalytic (enzyme) activity of the materials was examined by enzyme reaction. The enzyme activity was affected by the pore size and surface area, and the optimum pore diameter giving a high enzyme activity was 20 to 40m. The enzyme activity was also affected by irradiation temperature and addition concentration of crosslinking monomer. The heat and pH stability of the materials were higher than those of the native enzyme.     

C/C复合材料高温长寿命抗氧化复合涂层研究

采用包埋技术在C/C复合材料表面制备SiC-WSi₂/MoSi₂抗氧化复合涂层; 通过恒温氧化实验以及X射线衍射分析、扫描电镜观察及能谱分析, 研究了W、Mo含量对复合涂层微观结构和高温抗氧化性能的影响. 结果表明: 随着包埋粉料中W、Mo含量的增加, 所制备复合涂层的厚度先增加后减小; 含有10.0at％ W和Mo制备的复合涂层具有相对较大的厚度和较为致密的结构, 且WSi₂和MoSi₂含量相对较高; 氧化过程中在涂层表面形成致密和稳定的SiO₂玻璃保护膜; 在1500℃氧化315h后, 带有该涂层的C/C试样仍然没有失重, 且经过18次1500℃←→室温急冷急热后涂层没有开裂和脱落, 说明该涂层具有优异的抗氧化和抗热震性能.     

Characterization of functionalized nanoporous supports for protein confinement

Here we characterize a highly efficient approach for protein confinement and enzyme immobilization in NH(2)-?or HOOC-?functionalized mesoporous silica (FMS) with pore sizes as large as tens of nanometres. We observed a dramatic increase of enzyme loading in both enzyme activity and protein amount when using appropriate FMS in comparison with unfunctionalized mesoporous silica and normal porous silica. With different protein loading density in NH(2)-FMS, the negatively charged glucose oxidase (GOX) displayed an immobilization efficiency (I(e), the ratio of the specific activity of the immobilized enzyme to the specific activity of the free enzyme in stock solution) in a range from 30% to 160%, while the same charged glucose isomerase (GI) showed an I(e) of 100% to 120%, and the positively charged organophosphorus hydrolase (OPH) exhibited I(e) of more than 200% in HOOC-FMS. The enzyme-FMS composite was stained with the charged gold nanoparticles and imaged by transmission electron microscopy (TEM). Fourier transform infrared (FTIR) spectroscopy showed no major secondary structural change for the enzymes entrapped in FMS. Thanks to the large, rigid, open pore structure of FMS, the reaction rate and K(m) of the entrapped enzymes in FMS were comparable to those of the free enzymes in solution. In principle, the general approach described here should be applicable to many enzymes, proteins, and protein complexes since both pore sizes and functional groups of FMS are controllable.     

A high performance lipase preparation: characterization and atomic force microscopy

The goal of obtaining enzyme forms which show greater stability, higher catalytic efficiency, and reusability has been pursued since last several decades. Some novel biocatalyst designs have been evolved and protein coated micro-crystals (PCMCs) is one of them. Pseudomonas cepacia lipase coated micro-crystals were prepared by simultaneous precipitation of mixture of aqueous lipase solution and salts such as potassium sulphate by organic solvents. This resulted in lipase coated micro-crystals. The structures of micro-crystals were studied by atomic force microscopy (AFM). The AFM picture confirmed the enzyme coating over the potassium sulphate crystals. These PCMCs are in the size range of 500-1000 nm. These enzyme coated micro-crystals showed enhanced transesterification rates. Also, the PCMC were stable at 60 degrees C whereas the free enzyme lost all its activity. The enzyme coated micro-crystals prepared by 50 mg Pseudomonas cepacia lipase gave 96% conversion in 90 min whereas free enzyme gave 8% conversion. Even PCMCs prepared from 3.12 mg lipase gave 90% conversion in 10 h at 60 degrees C where as free lipase was inactive at 60 degrees C.     

聚丙烯亲水性微孔膜的制备及在锂电池中的应用

以聚丙烯接枝马来酸酐(PP-g-MAH)为改性剂与聚丙烯熔融共混,通过单向拉伸工艺制备亲水性聚丙烯微孔膜。使用差示扫描量热仪和红外光谱表征流延基膜的取向片晶结构,使用压汞仪和扫描电镜表征微孔膜的孔结构,使用Gurley值、水接触角和水蒸气透过率表征微孔膜的透过性和亲水性,将微孔膜组装成电池并测定电池的性能,研究PPg-MAH含量对微孔膜孔结构、亲水性、透过率及电池性能的影响。结果表明,PP-g-MAH共混含量为2.5%的微孔膜孔隙率和透气性得到提升,进一步增加PP-g-MAH含量会导致微孔膜孔结构变差,透过性能下降。微孔膜表面亲水性随PP-g-MAH含量增加持续改善,水蒸气透过率在PP-g-MAH含量为5%时达到最大值,但由于微孔膜孔结构在较高PPg-MAH含量下受到破坏,使得高PP-g-MAH含量下微孔膜水蒸气透过率下降。PP-g-MAH含量为2.5%和5%的微孔膜由于具有较好的孔结构和一定的电解液浸润性,使得锂电池的电荷转移电阻较低,首次充放电比容量较高。     

多孔青铜过滤片的制备工艺及结构特性探究

开发一种工艺简单、重复性好、孔形孔径易控制、制取成本低的铜基多孔材料制备工艺是当前的研究热点之一.本文以青铜粉为原料,K_2CO_3为造孔剂,采用烧结溶解法制备多孔青铜过滤片,研究了造孔剂、烧结温度对样品孔隙率的影响,分析了烧结温度、压制压力对样品最大孔径和透气系数的影响,以及孔隙率与抗压强度的关系.研究结果表明:当造孔剂体积分数为20%~40%时,所制备样品的孔隙率为22.8%~44.4%,开孔孔隙率为18.5%~37.2%;随着烧结温度的升高,样品孔隙率和透气系数下降;随着压制压力增加,最大孔径和透气系数均减小;随着样品孔隙率增大,抗压强度减小.当选择造孔剂体积分数30%、压制压力150 MPa、烧结温度800℃的工艺参数下,制备出孔隙率32.2%、最大孔径4.6μm、透气系数9.27 m~3/(h·k Pa·m~2)、压缩强度27.9 MPa的多孔青铜过滤片.     

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.     

Potentiometric and voltammetric investigations of H2/H+ catalysis by periplasmic hydrogenase from Desulfovibrio gigas immobilized at the electrode surface in an amphiphilic bilayer assembly.

Interactions of an enzyme with an organized amphipilic bilayer are explored as a general means of enzyme immobilization in electroenzymatic systems. Immobilization of Desulfovibrio gigas hydrogenase at the electrode surface involves hydrophobic interactions of the enzyme with the bilayer assembly consisting of octadecyltrichlorosilane and octadecylviologen (C18MV2+) molecules. Due to a hydrophobic character of the enzyme, these interactions direct the enzyme to occupy a central position in the bilayer's hydrocarbon region and lead to immobilization of 3 pmol/cm2 of the enzyme in the plane of the bilayer. This corresponds to 50% surface coverage. The immobilized enzyme catalyzes H2 oxidation mediated by the C18MW2+/.+ couple. This electroenzymatic scheme functions under steady-state voltammetric as well as potentiometric conditions in the pH range 3.5-10. Coupling of enzymatic activity to the electrode surface is accomplished via lateral diffusion of the octadecylviologen molecules along the bilayer assembly.     

A novel method for enzyme immobilization: direct encapsulation of acid phosphatase in nanoporous silica host materials

Immobilization of acid phosphatase (ACP) in mesoporous or, more generally, nanoporous silica has been accomplished via the sol-gel reactions of tetramethyl orthosilicate in the presence of ACP and of D-glucose (DG) as a nonsurfactant template, which is subsequently removed by water extraction after the formation of nanocomposite gels. Characterization of the silica host after the removal of DG shows that the pore size and volume generally increase with the DG content. At high DG contents, the silica hosts are nanoporous with interconnected nanoscaled pores/channels of regular diameter (e.g., 3.4 nm). Catalytic activity of ACP encapsulated in nanoporous hosts is significantly improved over that in microporous host prepared in the absence of DG. The apparent enzymatic activity at various pH values and substrate concentrations correlates well with the nanostructures of the host matrices. As the DG content is increased in the synthesis, the activity tends to increase. At a DG content of 42-60 wt%, the samples exhibit activities about triple that of the template-free control. These and other results from enzymatic kinetic studies suggest that the increase in the pore size and volume facilitates the transport of the substrate and product molecules in the host matrices, leading to the observed increase in activity. The thermal stability of ACP is remarkably improved upon immobilization. There is no detectable leakage of ACP from the host matrices and the biogels are reuseable. This study provides a useful protocol for the development of nanotechnology for various biocatalysts and biosensors.     

聚丙烯酸对纳米SiO_2薄膜性能的影响

采用溶胶-凝胶法,以正硅酸乙酯(TEOS)为前驱体、聚丙烯酸(PAA)为增孔剂制备大孔径SiO_2薄膜,研究了PAA添加量对薄膜孔径的影响、烧结温度与薄膜孔径及晶相转化之间的关系。通过热重分析仪(TG-DTG)、氮气吸附-脱附法、X射线衍射仪(XRD)、原子力显微镜(AFM)、红外光谱仪(IR)对样品性能进行表征。结果表明,随着PAA添加量的增多,孔径随之增大,当添加量达到15%(体积分数)时,薄膜表面出现裂纹;随着烧结温度的升高,SiO_2晶相发生转变及PAA完全炭化使得薄膜孔径增大。实验确定,PAA最佳加入量为10%,且最佳烧结温度为800℃时制得的SiO_2薄膜的热稳定性良好,表面平整致密,平均孔径为53.73nm。     

Hierarchical graphene foam-based phase change materials with enhanced thermal conductivity and shape stability for efficient solar-to-thermal energy conversion and storage

Recently,graphene foam (GF) with a three-dimensional (3D) interconnected network produced by template-directed chemical vapor deposition (CVD) has been used to prepare composite phase-change materials (PCMs) with enhanced thermal conductivity.However,the pore size of GF is as large as hundreds of micrometers,resulting in a remarkable thermal resistance for heat transfer from the PCM inside the large pores to the GF strut walls.In this study,a novel 3D hierarchical GF (HGF) is obtained by filling the pores of GF with hollow graphene networks.The HGF is then used to prepare a paraffin wax (PW)-based composite PCM.The thermal conductivity of the PW/HGF composite PCM is 87％ and 744％ higher than that of the PW/GF composite PCM and pure PW,respectively.The PW/HGF composite PCM also exhibits better shape stability than the PW/GF composite PCM,negligible change in the phase-change temperature,a high thermal energy storage density that is 95％ of pure PW,good thermal reliability,and chemical stability with cycling for 100 times.More importantly,PW/HGF composite PCM allows light-driven thermal energy storage with a high light-to-thermal energy conversion and storage efficiency,indicating its great potential for applications in solar-energy utilization and storage.