漆酶固定化及其在光纤氧传感器中的应用

以戊二醛作为交联剂,成功地将漆酶固定在四氨基酞菁铜(CuTAPc)-Fe3O4纳米复合粒子上.探讨了固定条件对固定化漆酶活力的影响,结果表明:当以10%的戊二醛与纳米载体交联8 h后,再以2g/L的漆酶溶液(pH=7)于4℃下固定所得固定化漆酶活力最高.固定化漆酶组装在光纤传感器上制备成性能良好的氧传感器.此传感器检测下限为0.45mg/L,精度为0.01 mg/L,响应时间小于30s.     

酶生物传感器固定化酶载体的研究

选用具有生物亲合性的蛋壳膜作载体,采用戊二醛为交联剂的交联法对葡萄糖氧化酶进行固定化,与用蛋壳膜作支撑体的溶胶-凝胶法固定化酶作比较.利用基于氧电极的葡萄糖氧化酶传感器模型,着重研究了不同条件下固定化酶的活性,发现蛋壳膜法固定化葡萄糖氧化酶的活性较高,证明了蛋壳膜是一种很好的固定化酶的载体材料.     

新型酶-微生物混合型传感器的研究

为了检测瓦斯中甲烷气体的浓度.在甲烷生物传感器基础上,利用甲烷单加氧酶在氧气中的催化特性,制备成固定化酶膜与甲烷氧化的微生物固定化层一起装在氧电极上,使传感器中催化剂的性能更好.分析了这种酶-微生物混合型传感器的工作原理、组成结构及微生物的固定方法.并通过实验证明这种传感器响应迅速,稳定性、重复性和精度较之传统的生物传感器有所提高.     

磁场对脲酶／葡萄糖氧化酶／α—淀粉酶催化活性的影响及磁场生物效应…

本文选择脲酶，葡萄糖氧化酶和α－淀粉酶为研究对象，分别以尼龙网和软聚氨酯泡沫为载体，以戊二醛为交联剂成功地研制了固定化酶柱，建立了简便，有效检测其溶解酶以及固定化酶催化活性的电导法和流动注射分光光度法，研究了固定化酶的性能及动力学参数，考察了不同条件下磁场对溶解酶以及固定化酶催化活性的影响。     

基于酶固定的新型抗坏血酸传感器的研究

以鸡蛋膜为基质,采用包埋一交联法,以戊二醛做交联剂,壳聚糖为包埋剂,固定抗坏血酸氧化酶,并偶联氧电极构建了抗坏血酸生物传感器。通过测定溶解氧浓度的变化对抗坏血酸的浓度进行测定。考察了溶液温度、pH、戊二醛浓度等因素对传感器响应行为的影响,优化的实验条件为：温度为室温,pH5．0,酶固定量为6Units,戊二醛质量分数为...     

葡萄糖氧化酶共价交联于鱼鳔膜、竹腔膜上的葡萄糖传感器的比较研究

以戊二醛作为交联剂,将葡萄糖氧化酶固定于生物载体--鱼鳔膜及竹腔膜上制成固定化酶.利用酶反应中氧对[Ru(dpp)3][(4-Clph)4B]2荧光猝灭制成荧光葡萄糖传感器.该文研究了这两种传感器的最佳测定条件、线性范围、重现性、选择性及稳定性,结果表明这两种生物支持体固定的葡萄糖酶传感器都具有良好的性能.     

碳纳米管-表面活性剂修饰的H2O2酶电极

酶在电极上的固定是酶传感器制备中的重要环节,它直接影响酶传感器的检测性能.该文利用静电吸附的自组装法以碳纳米管为载体将酶固定在电极上来制备酶传感器,该酶传感器制备过程简单、稳定性好、组装到电极表面的酶的量多并可保持其生物活性,可检测浓度在1.0×10-6～5.4×10-5mol/L范围内的H2O2,最低检测限为5.3×10-7mol/L.这种制备方法为检测农药残留生物传感器的制备提供了一种新的方法.     

新型脲酶场效应传感器的研制

酶半导体传感器(简称酶FET)是目前国内外研究得较多的生物敏半导体传感器,目前已研制出的主要有尿素、葡萄糖、青霉素、乙酰胆碱等酶FET。而酶的固定化方法则一般采用戊二醛交联法和光敏树脂法,用这些方法制得的脲酶FET,一般寿命在8～30d。我们采用尼龙膜固定脲酶,与场效应管结合,制得脲酶FET寿命可达60d以上,优于国内外文献报道值。     

脲酶电极的研究

前言脲酶电极是目前研究得较多的酶电极之一,一般是由适当的基础电极与固定化脲酶膜组合而成,作为基础电极主要有 NH_4~+离子电极,pH 电极,CO_2电极及氨气敏电极,而脲酶电极的固定化方法则曾用过聚丙烯酰胺包埋法,PVC(聚氯乙烯)包埋法,PVA(聚乙烯醇)包埋法及戊二醛交联法等。为了使该酶电极能更好地适应临床检验及食品分析的要求,并尽快将脲酶电极推广应用,我们对6种不同载体固定化方法,3种不同骨架电极及不同测试条件对电极性能的影响作了系统研究。结果表明,当采用化学交联法将脲酶固定在尼龙网上,用氨气敏电极作骨架电极时制得的脲酶电极性能最优;当选用 pH 为8.0～8.5的磷酸盐缓冲液时,电极在     

丙烯腈-丙烯酸共聚物多孔膜的硫化物传感器

采用自由基聚合方法在金电极表面合成丙烯腈-丙烯酸的共聚物多孔膜并用作固定酶的载体.再通过戊二醛将辣根过氧化物酶交联固定在丙烯腈-丙烯酸的共聚物多孔膜上制得过氧化氢生物传感器.硫化物抑制酶的活性使还原电流减小,根据电流域小的程度实现了对硫化物的测定.丙烯腈-丙烯酸共聚物多孔膜具有大的表面积和高的吸附能力.有利于改进传感器的检测下限.实验结果表明:将辣根过氧化物酶固定在丙烯腈-丙烯酸的共聚物多孔膜金电极上,作为一种检测硫化物的抑制型传感器,分析响应性能良好.     

Potentiometric urea biosensor based on BSA embedded surface modified polypyrrole film

A potentiometric biosensor based on bovine serum albumin (BSA) embedded surface modified polypyrrole has been developed for the quantitative estimation of urea in aqueous solution. The enzyme, urease (Urs), was covalently linked to free amino groups present over the BSA embedded modified surface of the conducting polypyrrole film electrochemically deposited onto an indium–tin-oxide (ITO) coated glass plate. The biosensor has been characterized by UV–visible, infrared spectroscopy and SEM. Potentiometric and spectrophotometric response of the enzyme electrode (Urs/BSA-PPy/ITO) were measured as a function of urea concentration in Tris–HCl buffer (pH 7.0). It has been found that the electrode responds to low urea concentration with wider range of detection. The electrode showed a linear response range of 6.6 × 10⁻⁶ to 7.5 × 10⁻⁴ M urea. The response time is about 70–90 s reaching to a 95% steady-state potential value and 75% of the enzyme activity is retained for about 2 months. These results indicate an efficient covalent linkage of enzyme to free amino groups of the BSA molecules over the surface of polypyrrole film, which leads to high enzyme loading, an increased lifetime stability of the electrode and an improved wide range of detection of low urea concentration in aqueous solution.     

Synthesis and performance of pH-sensitive hydrogel microspheres and in vitro evaluation as potential drug carriers

To accelerate the response rate of smart hydrogels to the environmental conditions, a novel pH-sensitive p(PEGMA-g-MAA) hydrogel microsphere with the controlled shapes and sizes were developed. Such monodispersed microspheres were synthesized via free radical polymerization under the protection of a multilayer stability system. The pH-responsibility of hydrogel microspheres was tested with the hydrogel bulk as a control. In vitro release studies were conducted in the simulated gastric fluid and intestinal juice with bovine serum albumin (BSA) as a model drug. The large specific surface areas endowed hydrogel microspheres a faster pH-responsibility than that of hydrogel bulk. In vitro release profiles showed that over 90 % BSA were released from hydrogel microspheres under the alkaline conditions (pH 7.4), which was faster than those from hydrogel bulks. In sum, the rapid pH-responsibility and ideal drug release profile could shorten the lag time to steady plasma-drug concentration, which was beneficial to increasing the therapeutic effect of the drugs.     

Aqueous two-phase extraction for bovine serum albumin (BSA) with co-laminar flow in a simple coaxial capillary microfluidic device

Microfluidic extraction based on a co-laminar flow of aqueous two-phase system is used to separate bovine serum albumin (BSA). Mass transfer between the continuous two-phase flows is demonstrated by the extraction of BSA in a microfluidic device. The protein concentrations of the BSA samples were determined using the Bradford method. Polyethylene glycol 4000 and ammonium sulfate ((NH₄)₂SO₄) served as model aqueous two-phase solutions. The appropriate flow rates of the aqueous two phases were thus determined. We can flexibly control the mass transfer area and time by simply adjusting the flow rate. It takes only 3.6 s for three extraction cycles in a coaxial microfluidic device to achieve a BSA recovery yield of 71.1 %, which is superior to the traditional beaker aqueous two-phase extraction process. In this study, co-laminar flow-based continuous microextraction is demonstrated and its mass transfer is analyzed by solving the diffusion model, based on a large specific interfacial area and surface renewal.     

Microfluidic system for electroelution of proteins from a clinical sampling strip

A clinical methylcellulose sampling strip is one of the popular means for collecting gingival crevicular fluid (GCF) from dental patients for dental disease diagnosis. In this research, a microfluidic device for protein elution from a sampling strip was fabricated with poly (dimethylsiloxane) (PDMS) polymer. Electoelution experiments were performed with fluorescein isothiocyanate (FITC) dye labeled bovine serum albumin (BSA) and ovalbumin (OVA). The total amount of eluted protein is measured by quantitative fluorescence imaging. About 50% of the initial concentration of BSA and OVA was eluted by the ~20 V/cm electric field. Electroelution is an appealing method for protein elution; however, the thickness of the wet strip (~400 μm) introduces interesting practical difficulties. During the electroelution process, unsteady electrokinetic phenomena by the pressure driven flow and the pH change of the reservoirs were observed. Several possible solutions to these problems are still under investigation including modifying reservoirs and thin polymer film coating of PDMS channel surfaces. This electroelution device would be a useful component of a fully integrated micro total analysis system for oral fluid samples.     

Laminated paper-based analytical devices (LPAD): fabrication, characterization, and assays

Paper-based microfluidic devices have recently garnered an increasing interest in the literature. The majority of these devices were produced by patterning hydrophobic zones in hydrophilic paper via photoresist or wax. Others were created by cutting paper using a laser. Here, we present a fabrication method for producing devices by simple craft-cutting and lamination, in a way similar to making an identification (ID) card. The method employs a digital craft cutter and roll laminator to produce laminated paper-based analytical devices (LPAD). Lamination with a plastic backing provides the mechanical strength for a paper device. The approach of using a craft cutter and laminator makes it possible to rapid-prototype LPAD with no more difficulty than producing a typical ID card, at very low cost. Devices constructed using this method have been exploited for simultaneous detection of bovine serum albumin (BSA) and glucose in synthetic urine with colorimetric assays. Both BSA and glucose are detectable at clinically relevant concentrations, with the detection limit at 2.5 μM for BSA and 0.5 mM for glucose.     

In situ modification of lipid-loaded MCM-41 channels with bovine serum albumin at a planar lipid bilayer for biosensing

An in situ method for modifying a receptor site on mesoporous silica MCM-41 channels in planar lipid bilayers is described, in which bovine serum albumin (BSA) is covalently linked to the MCM-41 channels via head groups of lipids loaded in the nanopores. Prior to receptor modification, lipid-loaded MCM-41 channels were incorporated with lipid bilayers formed at an aperture of a Teflon film. The in situ coupling of BSA to lipid-loaded MCM-41 channels at the lipid bilayer interface was achieved by the sulfhydryl coupling method. The lipid bilayers containing BSA-modified MCM-41 exhibited channel-like currents, which were augmented in a concentration-dependent manner by the addition of anti-BSA at fM level. The in situ modification of lipid-loaded MCM-41 channels with BSA by the amine coupling technique was also investigated. The potential of the present approach for the development of channel-type biosensors is discussed in terms of modifying bilayer interfaces with bioreceptors.     

Localized protein immobilization on microstructured polymeric surfaces for diagnostic applications

We demonstrate a localized protein immobilization method based on controlled physical adsorption on the three-phase boundary of an aqueous phase, a gas phase, and a polymeric material. By imprinting micrometer and sub-micrometer pillars onto a polymeric foil, superhydrophobic surfaces are fabricated. Those structures force the fluid locally into the Cassie–Baxter state and generate an artificial three-phase boundary at the edges of the imprinted pillars. First, fluorescence-labeled bovine serum albumin (BSA) and streptavidin dissolved in various buffer solutions are utilized to investigate protein adsorption on the structured surfaces. A stable adsorption of the respective protein on the three-phase boundary is observed. The following experiments use streptavidin adsorbed on the pillars to immobilize biotinylated antibodies for analyte detection. The pillars are passivated with an excess concentration of BSA to reduce nonspecific protein adsorption. Implemented in a lab-on-a-chip device, the proposed immobilization method is utilized in a sandwich assay to detect the inflammation marker C-reactive protein in human serum, showing the potential of this immobilization method for diagnostic applications. The method overcomes laborious procedures to immobilize proteins on thermoplastic materials, which enables the fast transfer of point-of-care applications from research to commercial scale.     

Application of functionalised carbon nanotubes immobilised into chitosan films in amperometric enzyme biosensors

A new approach for building a bio-conductive interface for enzyme immobilisation is described. This strategy permits very simple preparation of the enzyme biosensor and also reveals direct electron transfer features. A graphite-epoxy resin composite (GrEC) electrode modified with functionalised multi-wall carbon nanotubes (MWCNTs) immobilised by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide together with N-hydroxysuccinimide (EDC–NHS) in a chitosan (Chit) matrix was prepared and characterised by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in the presence of hexaammineruthenium (III) chloride. It was then used as a base for glucose oxidase (GOx) immobilisation by the simple method of crosslinking with glutaraldehyde (GA) with bovine serum albumin (BSA) as carrier protein. The resulting mediator-free biosensor was applied to the determination of glucose in amperometric mode at different applied potentials and the mechanism of reaction was also investigated by cyclic voltammetry, with and without dissolved oxygen in solution. Analytical parameters, as well as reproducibility, repeatability and stability were determined. Interferences were assessed using different compounds usually present in natural samples, such as wines, juices or blood, in order to evaluate the selectivity of the developed biosensor. The novel combination of carbon nanotubes immobilised with chitosan crosslinked with EDC–NHS and glucose oxidase immobilised by crosslinking with glutaraldehyde offers an excellent, easy to make biosensor for glucose determination without interferences.     

pH型有机磷水解酶生物传感器的稳态模型

从分析pH型有机磷水解酶(OPH)生物传感器(简称OPH-pH型传感器)的工作原理出发,建立了描述稳态检测过程的扩散传质方程组,并根据方程组的特点将二阶微分方程组简化为二阶微分方程和非线性方程的求解,从而建立了OPH-pH型传感器的稳态模型。模型的计算结果给出了传感器性能的影响因素,可对传感器的设计提供一定的理论指导,自行制备的传感器实验结果与模型的计算结果基本吻合,验证了模型的正确性。     

用直接实验设计方法优化聚丙烯腈基膜的制备条件

以聚丙烯腈为聚合物，磷酸或聚乙二醇-600为添加剂，N-甲基吡咯烷酮为溶剂，采用L-S相转换法制备PAN超滤膜。利用计算机直接实验设计方法设计制膜的配方，用Statistic Analytic System(SAS)统计软件对膜的水通量、牛血清蛋白截留率和平均孔径进行回归分析，得出影响PAN基底膜性能的主要因素，并且优化了PAN基膜制备的工艺条件。实验结果表明，PAN浓度、添加剂种类和添加剂浓度是PAN基膜性能的主要影响因素；在一定的浓度范围内，以适当的PAN浓度和添加剂浓度均可制备出渗透性能较好的PAN超滤膜；SAS统计软件对PAN基膜的截留率和平均孔径的预测值与实验值吻合较好。