基于纳米金的比色法在生理活性分子分析中的应用进展

以纳米金或功能化纳米金为探针分子发展起来的比色法,因其具有肉眼可视等诸多优势,已广泛应用于生理活性物质的检测分析中。主要综述了纳米金比色法的应用原理及其在生理小分子分析方面的最新进展,并对基于纳米金的比色法今后的发展趋势进行了展望。     

多菌灵的紫外分光光度法分析

提出在波长281纳米处用紫外分光比色法测定多菌灵,此法可克服非水滴定法邻苯二胺干扰。相对误差小于0.5%,标准偏差为0.37%。     

基于纳米金的汞离子传感器研究进展

综述了近年来基于纳米金的Hg2+传感器研究进展,包括比色法、荧光法、电化学法和表面增强拉曼散射(SERS)法,详述了各种传感器的机理及优缺点,并对未来的挑战和发展机遇进行了展望。     

纳米二氧化钛对隔离子的吸附研究

研究了纳米二氧化钛吸附溶液中镉离子的有效方法和途径.采用双硫腙直接比色法测定镉离子浓度,详细研究了纳米二氧化钛吸附镉离子过程中的影响因素.通过实验,确定了吸附的最佳条件,进一步研究了吸附镉离子后二氧化钛的洗脱以及设想模拟实体水样中痕量镉离子的检测.样品体系在pH=10,震荡时间9 min以上的条件下,纳米二氧化钛对镉离子的吸附率可达99 %.吸附在纳米二氧化钛上的镉离子可以用0.1 mol/L的硝酸进行洗脱,洗脱率在95 %以上.     

二氧化锰纳米颗粒比色法测定谷胱甘肽

基于二氧化锰纳米颗粒具有优异的模拟酶特性,能够高效地催化氧化3,3',5,5'-四甲基联苯胺产生明显的蓝色变化,而谷胱甘肽的加入能够引起二氧化锰的降解从而抑制了基于二氧化锰的显色反应。据此制备了二氧化锰纳米颗粒并且建立了测定谷胱甘肽的比色法,结果表明,在最佳条件下,谷胱甘肽的线性范围为8～70μmol/L,并且表现出了良好的选择性。     

Fe3O4磁性纳米复合材料的制备及载药性能

利用聚乙二醇水溶性高及良好的生物相容性等特性对Fe₃O₄磁性纳米粒子进行表面改性,从而改善Fe₃O₄易于团聚的缺陷。通过一步法制备具有核壳结构的聚乙二醇/Fe₃O₄磁性纳米复合材料,使用FTIR、XRD、TEM和VSM对复合纳米材料的结构、形貌和性能进行了表征。采用超声法合成了阿霉素聚乙二醇修饰的Fe₃O₄磁性纳米包合物,用光度法对磁性纳米复合物的包封率进行了系统的分析,平均包封率为54.83%,并通过四唑盐(MTT)比色法证明了包合物对K150细胞有明显的抑制作用。研究结果表明:合成的磁性纳米复合材料拥有良好的形貌和载药性能,可作为一种新型的药物载体以达到靶向运输的效果,从而提高药物的生物利用度。     

核壳结构磁性纳米复合物的合成及载药性能

利用化学交联法合成了壳聚糖改性的四氧化三铁磁性纳米复合物。通过透射电子显微镜（TEM）、傅里叶转换红外光谱（FTIR）、粒度分析仪（Nano-ZS）和振动样品磁强计（VSM）对该复合物的形貌、粒径、物相组成及磁性能进行了表征分析。表明该磁性纳米复合物具有核壳结构。再用超声法合成了阿霉素-壳聚糖修饰的磁性纳米包合物,用紫外-可见（UV-Vis）分光光度计检测了该复合物的包封率,平均值达到46.13%。通过四唑盐（MTT）比色法证明了包合物对K150细胞的生物抑制作用。以上结果表明,该磁性纳米复合物具有良好的生物相容性和载药活性。     

基于贵金属纳米粒子比色检测法在食品检测中的应用

民以食为天,食品安全一直是人们关注的焦点,也是民生保障的重中之重。对食品进行安全评价则需要定性定量地分析出食品营养成分或有害物质的含量。目前常用的检测方法主要是液相色谱法,气相色谱法,电化学分析等仪器分析法以及其联用技术。这些方法却存在着设备昂贵,操作繁琐,检测时间长等缺点,不能满足现场即时检测的要求。贵金属纳米粒子比色法具有灵敏度高、选择性好、操作简单、反应快速等优点,并可以开发出检测试剂盒,有一定的商业潜力,得到了人们的广泛研究与应用。本文列举近几年基于贵金属纳米粒子比色在食品安全上的一些主要研究工作。     

纳米羟基磷灰石的表征及其对肝癌抑制作用的研究

利用化学方法辅助超声制备出稳定单分散的羟基磷灰石(HAP)纳米粒子体系.应用原子力显微镜(AFM)、粒径电位仪对HAP纳米粒子进行表征;采用形态学观察和MTT比色法研究HAP纳米粒子对细胞系Bel-7402人肝癌细胞的作用;从单细胞荧光元素微区分析、超微结构及细胞周期的改变研究其作用机理.实验结果表明:HAP纳米粒子在体外对Bel-7402人肝癌细胞具有明显的抑制作用,通过进入到癌细胞内,阻滞细胞周期的进展,使细胞在G1期堆积,导致癌细胞胀亡.     

核壳结构磁性纳米复合物的合成及载药性能

利用化学交联法合成了壳聚糖改性的四氧化三铁磁性纳米复合物。通过透射电子显微镜(TEM)、傅里叶转换红外光谱(FTIR)、粒度分析仪(Nano-ZS)和振动样品磁强计(VSM)对该复合物的形貌、粒径、物相组成及磁性能进行了表征分析。表明该磁性纳米复合物具有核壳结构。再用超声法合成了阿霉素-壳聚糖修饰的磁性纳米包合物,用紫外-可见(UV-Vis)分光光度计检测了该复合物的包封率,平均值达到46.13%。通过四唑盐(MTT)比色法证明了包合物对K150细胞的生物抑制作用。以上结果表明,该磁性纳米复合物具有良好的生物相容性和载药活性。     

磁响应交联糖化酶聚集体的制备及催化特性

提出了一种采用羧基磁性纳米粒子制备杂化磁响应交联酶聚集体（M-CLEAs）的方法。表面羧基修饰的约10 nm的磁性纳米粒子与酶分子表面的氨基位点通过静电相互作用,形成复合物,在磁场作用下可将磁性纳米粒子-酶复合物从溶液中分离,经戊二醛交联即形成M-CLEAs。传统的表面氨基修饰的磁性纳米粒子与酶需在沉淀剂作用下,从溶液中分离,而后采用戊二醛共交联,而本方法无须沉淀剂,过程更为简化。以糖化酶为对象,对该过程的影响因素（交联时间、pH、酶浓度、戊二醛浓度等条件）进行了探索,并对制得的M-CLEAs的酶学性质进行了较为详细考察。结果表明,最优制备条件为：酶浓度1 mg·ml^-1,磁流体浓度10 mg·ml^-1,戊二醛浓度0.25%（质量体积比）,在pH 6.0下交联反应6 h,最终载酶量可达80 mg·g^-1、比活为50 U·mg^-1。制得的固定化酶pH稳定性、热稳定性和储存稳定性均显著改善,可实现糖化酶重复使用10次,仍保留接近60%的酶活。     

Improvement of chymotrypsin enzyme stability as single enzyme nanoparticles

Individual enzyme molecules were coated with a nanometer-scale polymer network in order to increase their stability, providing longer lifetime of enzymes for biochemical processes. This polymer nanolayer is thin and porous enough [Kim, J., Grate, J.W., 2003. Single-enzyme nanoparticles armored by a nanometer-scale organic/inorganic network. Nano Letters 3, 1219-1222] to allow practically unhindered diffusion of the substrate from the solution to the active site of the enzyme, and unhindered transfer of the product from the active site to the solution. During the three-step preparation of the individual enzyme nanoparticles, the chymotrypsin enzyme investigated can lose 30-50% of its original activity. The main cause for a decrease in the enzyme's activity is the UV-light irradiation used during the polymerization step of the pretreatment. The UV-light can destroy the tertiary structure of the enzyme, and this can lead to reduced activity. The enzyme modification and solubilization steps of the treatment methodology do not essentially lower enzyme activity. The morphology and size of enzyme nanoparticles prepared was examined by transmission electron microscopy and demonstrated in this paper. The activity's change of the free and the covered enzyme was investigated at different temperatures, shaking frequencies and pH values. It has been proved that the preparation of enzyme nanoparticles can essentially stabilize the enzyme. Its activity changes much slower than that of native enzyme. The pretreated enzyme can have relatively high residual activity under extreme pH values and temperature, as well. The essence of the results presented is that stability of the enzyme can be increased significantly by covering individual enzymes with a thin, porous polymer layer.     

仿生合成氧化锆固定化漆酶处理孔雀石绿

以溶菌酶作为诱导剂,仿生合成了ZrO₂固定化漆酶纳米颗粒,其酶活回收率达59%,采用场发射扫描电子显微镜（FESEM）、能谱仪（EDS）、热重分析仪（TGA）等手段对ZrO₂纳米颗粒及ZrO₂固定化漆酶颗粒进行表征,结果表明漆酶可成功固定到ZrO₂颗粒中,同时还证明了溶菌酶既作为诱导剂催化ZrO₂的形成,又作为生物模板同酶一起包埋在ZrO₂颗粒中。固定化漆酶的最适pH为3,最适温度为70℃,相比于游离酶,其pH、温度稳定性都有明显提高;固定化漆酶纳米颗粒在4℃下储存30d,活性为初始酶活的95%,重复使用5次,固定化酶的残余酶活力仍有60%。此外,固定化漆酶在6h内对孔雀石绿染料的脱色率高达95%以上,通过紫外-可见吸收光谱分析（UV-vis）可知,固定化漆酶对孔雀石绿染料的处理是由吸附和降解联合作用引起的脱色。     

Regulation of Enzyme Activity through Interactions with Nanoparticles

The structure and function of an enzyme can be altered by nanoparticles (NPs). The interaction between enzyme and NPs is governed by the key properties of NPs, such as structure, size, surface chemistry, charge and surface shape. Recent representative studies on the NP-enzyme interactions and the regulation of enzyme activity by NPs with different size, composition and surface modification are reviewed.     

纳米粒子固定化双酶耦合体系连续催化制备(R)-苯基乙二醇

将活化醇盐水解法制备的SiO2纳米粒子分别与羰基还原酶(CR)和甲酸脱氢酶(FDH)进行共价固定化,固定化CR与FDH耦合,连续催化转化b-羟基苯乙酮制备(R)-苯基乙二醇,考察了NADH的再生与循环利用性. 结果表明,纳米粒子固定化CR和FDH酶载量分别为3.32和5.55 mg/g,催化活性为游离酶的50%~60%,最适反应pH值分别为6.5和8.5,最适反应温度分别为40和45℃. 耦合体系进行12批次反应,产物(R)-苯基乙二醇累积量达35.6 g/L,纳米粒子生产能力达178 g/g. 纳米粒子固定化酶经简单离心收集后可重复利用.     

含超细Au颗粒的乳酸氧化酶和葡萄糖氧化酶生物传感器

制备了小于10 nm的金颗粒，并利用其进行固定化葡萄糖氧化酶和乳酸氧化酶的研究. 实验发现，金纳米颗粒可以大幅度提高葡萄糖氧化酶电极和乳酸氧化酶电极的电流响应，响应电流从相应浓度的几十纳安增强到几千纳安. 探讨了金纳米颗粒在固定化酶中所起的作用.     

Preparation of chitosan‐coated magnetite nanoparticles and application for immobilization of laccase

In this study, immobilization of laccase (L) enzyme on magnetite (Fe₃O₄) nanoparticles was achieved, so that the immobilized enzyme could be used repeatedly. For this purpose, Fe₃O₄ nanoparticles were coated and functionalized with chitosan (CS) and laccase from Trametes versicolor was immobilized onto chitosan‐coated magnetic nanoparticles (Fe₃O₄‐CS) by adsorption or covalent binding after activating the hydroxyl groups of chitosan with carbodiimide (EDAC) or cyanuric chloride (CC). For chitosan‐coated magnetic nanoparticles, the thickness of CS layer was estimated as 1.0–4.8 nm by TEM, isoelectric point was detected as 6.86 by zeta (ζ)‐potential measurements, and the saturation magnetization was determined as 25.2 emu g^?1 by VSM, indicating that these nanoparticles were almost superparamagnetic. For free laccase and immobilized laccase systems, the optimum pH, temperature, and kinetic parameters were investigated; and the change of the activity against repeated use of the immobilized systems were examined. The results indicated that all immobilized systems retained more than 71% of their initial activity at the end of 30 batch uses. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

新型层状交联酶聚集体的制备条件优化与性质研究

以假丝酵母脂肪酶（Candida sp.lipase）为研究对象,开发了一种新型层状交联酶聚集体。用蛋白或氨基酸对纳米氧化锌粒子进行修饰,继以交联剂交联后作为核芯,酶分子再交联在纳米核表面形成层状结构。实验结果表明牛血清白蛋白（BSA）是纳米氧化锌适宜的修饰剂。并且对纳米芯层状交联酶聚集体（BSA-N-LCLEAs）其他制备条件进行了优化,优化后BSA-N-LCLEAs制备条件为：沉淀剂硫酸铵饱和度为58%,交联剂戊二醛浓度为3.5%,交联温度和时间分别为0℃和2 h。BSA-N-LCLEAs酶活收率较传统CLEAs提高了196.5%。扫描电镜表征表明BSA-N-LCLEAs较传统CLEAs孔道大幅增加。纳米芯层状CLEAs的pH稳定性和热稳定性也都比传统CLEAs有所提高,并将该固定化酶用于催化维生素E琥珀酸酯的合成,反应五批次后反应产率还能达90%左右,说明该新型交联酶聚集体具有良好的催化活性和操作稳定性。     

Enzyme–magnetic nanoparticle hybrids: new effective catalysts for the production of high value chemicals

Magnetic nanoparticles (5–100 nm in diameter) offer the science community a new dimension in many key areas of research, notably nano‐medicine. However, use as catalyst support material (including enzyme immobilization) has largely been overlooked despite the benefits of a facile catalyst separation post‐reaction with reuse through the application of an external magnetic field. This is largely due to the high cost associated with magnetic nanoparticle synthesis in addition to enzyme operating costs. This can, however, be compensated for by effective catalyst recycling in the production of high value chemicals, particularly chiral drugs and their intermediates, circumventing laborious multi‐stage separation and purification procedures in conventional synthesis. Indeed, enzymes (such as lipase) on magnetic carriers have shown prolonged activity in organic solvents, even after recycling, when compared with free enzymes. Further developments can be directed at multi‐functional magnetic nanoparticles with the creation of a reactant‐specific micro‐environment to facilitate multi‐stepped reactions. The potential of enzyme‐magnetic nanoparticle hybrids is discussed and applications in the synthesis of fine chemicals, including precursors for drugs, are identified. Copyright © 2012 Society of Chemical Industry     

Nanostructures for enzyme stabilization

Recent breakthroughs in nanotechnology have made various nanostructured materials more affordable for a broader range of applications. Although we are still at the beginning of exploring the use of these materials for biocatalysis, various nanostructures have been examined as hosts for enzyme immobilization via approaches including enzyme adsorption, covalent attachment, enzyme encapsulation, and sophisticated combinations of methods. This review discusses the stabilization mechanisms behind these diverse approaches; such as confinement, pore size and volume, charge interaction, hydrophobic interaction, and multipoint attachment. In particular, we will review recently reported approaches to improve the enzyme stability in various nanostructures such as nanoparticles, nanofibers, mesoporous materials, and single enzyme nanoparticles (SENs). In the form of SENs, each enzyme molecule is surrounded with a nanometer scale network, resulting in stabilization of enzyme activity without any serious limitation for the substrate transfer from solution to the active site. SENs can be further immobilized into mesoporous silica with a large surface area, providing a hierarchical approach for stable, immobilized enzyme systems for various applications, such as bioconversion, bioremediation, and biosensors.