固定化多酶策略及易分离的载体材料研究进展

多酶级联反应是现代工业过程中重要的生物技术。然而,酶的分离和回收是一项繁琐而费力的工作,因此酶的固定化是实际应用中的关键问题。固定化多酶可以通过底物通道提高酶的催化活性,而易分离的载体材料有利于酶的稳定性和易于回收再利用。本文综述了近年来固定化多酶策略及易分离的载体材料相关研究,内容包括不同固定策略的多酶复合体,阐述了适合固定化酶的易于分离的载体材料,特别是磁性纳米颗粒和膜状材料无需离心即可从本体溶液中分离。总结了固定化多酶在食品生产和生物传感器领域的实际应用,最后对固定化多酶催化反应的发展前景和趋势进行了展望。     

Glucose oxidase–magnetite nanoparticle bioconjugate for glucose sensing

Immobilization of bioactive molecules on the surface of magnetic nanoparticles is of great interest, because the magnetic properties of these bioconjugates promise to greatly improve the delivery and recovery of biomolecules in biomedical applications. Here we present the preparation and functionalization of magnetite (Fe₃O₄) nanoparticles 20 nm in diameter and the successful covalent conjugation of the enzyme glucose oxidase to the amino-modified nanoparticle surface. Functionalization of the magnetic nanoparticle surface with amino groups greatly increased the amount and activity of the immobilized enzyme compared with immobilization procedures involving physical adsorption. The enzymatic activity of the glucose oxidase-coated magnetic nanoparticles was investigated by monitoring oxygen consumption during the enzymatic oxidation of glucose using a ruthenium phenanthroline fluorescent complex for oxygen sensing. The glucose oxidase-coated magnetite nanoparticles could function as nanometric glucose sensors in glucose solutions of concentrations up to 20 mmol L^–1. Immobilization of glucose oxidase on the nanoparticles also increased the stability of the enzyme. When stored at 4°C the nanoparticle suspensions maintained their bioactivity for up to 3 months.     

Colloidal stable silica encapsulated nano-magnetic composite as a novel bio-catalyst carrier

A colloidal stable silica-encapsulated magnetic nano-composite of a controlled dimension is, for the first time, employed to carry beta-lactamase via chemical linkage on the silica overlayer: activity study reflects that this new type of immobilisation allows site (enzyme) isolation, accessibility as good as free enzyme and recovery & reusability upon application of magnetic separation.     

磁性二氧化硅微球的表面修饰及其在植物基因组核酸纯化中的应用

在利用自主研发的专利技术制备球形磁性硅胶微球的基础上,对磁性硅胶微球进行表面改性,使其表面分别键合硅羟基、环氧基、邻二醇基和羧基等官能团,并对表面官能团进行了定量研究。以小牛胸腺基因组脱氧核糖核酸(DNA)为模型化合物,研究了核酸在不同表面官能团的磁性硅胶上的吸附和脱附行为,发现表面具有硅醇基的磁球对DNA的回收率最高。将改性后磁性微球应用于玉米DNA的提取,得到了平均长度大于8kb的高纯度基因组DNA。与传统的有机溶剂抽提法相比,基于磁性微球的核酸固相萃取法具有快速简便、省时省力、易于自动化的特点,适合于大规模植物基因组DNA的样品制备。     

Methodology for determination of magnetic force of polymeric nanocomposites

Magnetic nanocomposites present several interesting uses. They are very useful in environmental recovery, drug delivery and sensor applications. However, sophisticated magnetic measurements are very complex and present high costs, which may sometimes prevent research on these materials. Therefore, this paper presents a magnetic force test, which can be performed at relatively low cost and produces interesting results, which are very useful to support the development of these magnetic materials. Specifically, polylactic acid (PLA)/maghemite nanocomposites were prepared and characterized using Fourier transform infrared (FTIR) spectroscopy, wide-angle X-ray scattering (WAXS), small-angle X-ray scattering (SAXS) and size-exclusion chromatography (SEC). Our results demonstrate that nanocomposites, were obtained, which were also subjected to the magnetic force and magnetic susceptibility tests. The results of these latter tests were found to be linearly related, which proves the utility of the magnetic force test as a practical characterization technique.     

Improved Thermal and Reusability Properties of Xylanase by Genipin Cross-Linking to Magnetic Chitosan Particles

Enzymes are gradually increasingly preferred over chemical processes, but commercial enzyme applications remain limited due to their low stability and low product recovery, so the application of an immobilization technique is required for repeated use. The aims of this work were to produce stable enzyme complexes of cross-linked xylanase on magnetic chitosan, to describe some characteristics of these complexes, and to evaluate the thermal stability of the immobilized enzyme and its reusability. A xylanase was cross-linked to magnetite particles prepared by in situ co-precipitation of iron salts in a chitosan template. The effect of temperature, pH, kinetic parameters, and reusability on free and immobilized xylanase was evaluated. Magnetization, morphology, size, structural change, and thermal behavior of immobilized enzyme were described. 1.0?±?0.1 μg of xylanase was immobilized per milligram of superparamagnetic chitosan nanoparticles via covalent bonds formed with genipin. Immobilized xylanase showed thermal, pH, and catalytic velocity improvement compared to the free enzyme and can be reused three times. Heterogeneous aggregates of 254 nm were obtained after enzyme immobilization. The immobilization protocol used in this work was successful in retaining enzyme thermal stability and could be important in using natural compounds such as Fe₃O₄@Chitosan@Xylanase in the harsh temperature condition of relevant industries.

     

Enzyme shadowing: using antibody-enzyme dually-labeled magnetic particles for fast bacterial detection

Using magnetic particles and immunoseparation for target recovery and detection has been reported to improve the performance and detection limits of traditional analytical methods. For example, magnetic immunocapture can be coupled to detection in a sandwich format using an antibody (Ab) labeled with a reporter molecule or enzyme. In this work we demonstrate that simultaneous incorporation of capture and reporter biocomponents onto the sensing surface is possible and provides assays that are extremely fast and easy to carry out. As a proof of concept, we have produced dually-labeled magnetic particles, simultaneously functionalized with antibody and reporter enzyme. Subsequent capture of Escherichia coli generates a shadowing effect on the particle surface and interferes with the activity of a number of enzyme units. The decrease in signal recorded is proportional to the bacterial concentration and is specific for the target microorganism, with a detection limit of 10(3)-10(4) cell mL(-1) in an assay of about one hour.     

磁性高分子微球用于固定化酶的研究进展

磁性高分子微球是近20年来发展起来的一种新型功能高分子材料,并已在生物化工、细胞学、生物医学工程等领域得到了广泛应用.本文主要介绍磁性高分子微球的制备和性质以及在固定化酶中的应用.     

磁性高分子微球用于固定化酶的研究进展生物材料结构及性质的基础研究及其应用于药物的控制释放

磁性高分子微球是近20年来发展起来的一种新型功能高分子材料,并已在生物化工、细胞学、生物医学工程等领域得到了广泛应用。本文主要介绍磁性高分子微球的制备和性质以及在固定化酶中的应用。     

Modification and characterization of artificially patterned enzymatically active surfaces by scanning electrochemical microscopy

This review summarizes the characterization of localized enzymatic activity by scanning electrochemical microscopy (SECM). After introducing the concepts of feedback imaging and generator-collector experiments with enzyme-modified solid surfaces, a comparison of the merits and limitations of both approaches is given and further illustrated by selected applications. They include enzyme-modified patterned monolayers, enzyme-modified polymer microstructures and enzyme-modified metal microstructures. Such configurations are important for the development of miniaturized bioanalytical systems with proteins, such as miniaturized enzyme electrode arrays. SECM has emerged as an ideal tool for prototyping of such systems. It also offers several mechanisms for local surface modifications under conditions compatible with conservation of protein functionality of enzymes and antibodies. The subsequent imaging of the immobilized activity provides direct information about local immobilized enzyme activity. The range of biotechnological applications can be expanded by labeling other biomolecules, such as monoclonal antibodies, with appropriate enzymes. Miniaturized electrochemical enzyme immunoassays that apply the sandwich format and SECM as the detection method are reviewed. They have been performed on microstructured supports after reagent spotting or on agglomerates of surface-modified magnetic microbeads. Finally, current challenges are listed with indications of ongoing research to overcome current limitations by means of instrumental improvements.     

Composite magnetic particles as carriers for laccase from Trametes versicolor

In this paper we report a study of laccase immobilisation on different kinds of carrier particles. The immobilisation of enzyme on the particle surface with respect to the immobilisation efficiency and the properties of the immobilised enzymes is discussed. The immobilisation of laccase on polystyrene particles bearing reactive beta-diketone groups is characterised by high efficiency, but grafting of the enzyme increases the stability of the colloidal system, which makes the separation/purification procedure difficult. Additionally, the extreme colloidal stability of the immobilisates hinders the application of such particles with immobilised enzymes in some applications where the recycling of the enzyme should be performed. It has been found that hybrid PS-AAEM particles equipped with maghemite show similar immobilisation efficiency to that of their analogues without maghemite and can additionally be manipulated in magnetic fields. The activity of the immobilised laccase is much higher in the pH region 5-7 and the temperature range 50-70 degrees C as compared with that of the free enzyme. Immobilised enzymes also exhibit much better storage stability.     

Magnetic porous carbon nanocomposites derived from metal-organic frameworks as a sensing platform for DNA fluorescent detection

Metal-organic frameworks (MOFs) have emerged as very fascinating functional materials due to their tunable nature and diverse applications. In this work, we prepared a magnetic porous carbon (MPC) nanocomposite by employing iron-containing MOFs (MIL-88A) as precursors through a one-pot thermolysis method. It was found that the MPC can absorb selectively single-stranded DNA (ssDNA) probe to form MPC/ssDNA complex and subsequently quench the labelled fluorescent dye of the ssDNA probe, which is resulted from the synergetic effect of magnetic nanoparticles and carbon matrix. Upon the addition of complementary target DNA, however, the absorbed ssDNA probe could be released from MPC surface by forming double-stranded DNA with target DNA, and accompanied by the recovery of the fluorescence of ssDNA probe. Based on these findings, a sensing platform with low background signal for DNA fluorescent detection was developed. The proposed sensing platform exhibits high sensitivity with detection limit of 1 nM and excellent selectivity to specific target DNA, even single-base mismatched nucleotide can be distinguished. We envision that the presented study would provide a new perspective on the potential applications of MOF-derived nanocomposites in biomedical fields.     

Rapid automatic nucleic acid purification system based on gas–liquid immiscible phase

The continuous expansion of nucleic acid detection applications has resulted in constant developments in rapid, low-consumption, and highly automated nucleic acid extraction methods. Nucleic acid extraction using magnetic beads across an immiscible phase interface offers significant simplification and parallelization potential. The gas–liquid immiscible phase valve eliminates the requirement for complicated cassettes and is suitable for automation applications. By analyzing the process of magnetic beads crossing the gas-liquid interface, we utilized a low magnetic field strength to drive large magnetic bead packages to cross the gas-liquid interface, providing a solution of high magnetic bead recovery rate for solid-phase extraction with a low-surfactant system based on gas-liquid immiscible phase valve. The recovery rate of magnetic beads was further improved to 90%–95% and the carryover of the reagents was below 1%. Consequently, a chip and an automatic system were developed to verify the applicability of this method for nucleic acid extraction. The Hepatitis B virus serum standard was used for the extraction test. The extraction of four samples was performed within 7 minutes, with nucleic acid recovery maintained above 80% and good purity. Thus, through analysis and experiments, a fast, highly automated, and low-consumption nucleic acid recovery method was proposed in this study.     

Modification and characterization of artificially patterned enzymatically active surfaces by scanning electrochemical microscopy

This review summarizes the characterization of localized enzymatic activity by scanning electrochemical microscopy (SECM). After introducing the concepts of feedback imaging and generator-collector experiments with enzyme-modified solid surfaces, a comparison of the merits and limitations of both approaches is given and further illustrated by selected applications. They include enzyme-modified patterned monolayers, enzyme-modified polymer microstructures and enzyme-modified metal microstructures. Such configurations are important for the development of miniaturized bioanalytical systems with proteins, such as miniaturized enzyme electrode arrays. SECM has emerged as an ideal tool for prototyping of such systems. It also offers several mechanisms for local surface modifications under conditions compatible with conservation of protein functionality of enzymes and antibodies. The subsequent imaging of the immobilized activity provides direct information about local immobilized enzyme activity. The range of biotechnological applications can be expanded by labeling other biomolecules, such as monoclonal antibodies, with appropriate enzymes. Miniaturized electrochemical enzyme immunoassays that apply the sandwich format and SECM as the detection method are reviewed. They have been performed on microstructured supports after reagent spotting or on agglomerates of surface-modified magnetic microbeads. Finally, current challenges are listed with indications of ongoing research to overcome current limitations by means of instrumental improvements. Received: 19 December 2000 / Revised: 26 February 2001 / Accepted: 2 March 2001     

Recent Developments and Applications of Three-Phase Partitioning for the Recovery of Proteins

Three-phase partitioning (TPP) is rapidly developing as a novel bioseparation technique for the separation and purification of biomolecules. TPP has been applied in a wide range of applications including enzyme stability and enhancement of its catalytic activity. The partitioning into three phases is mainly dependent on the concentration of alcohol and salt used. TPP provides high enzyme recovery and can be utilized along with external techniques such as ultrasound, microwave assisted, microaffinity ligand-facilitated and also ionic-liquid based. This technique has attracted interest in the large scale recovery of proteins from crude feedstocks or fermentation broths. In this review, the basic principles, refolding of proteins using TPP, key design variables of TPP, types of TPP, applications of TPP in food industry as well as the challenge of TPP were analyzed. The work presented in this review will be beneficial for further researches in TPP or related separation techniques.     

Continuous microfluidic DNA extraction using phase-transfer magnetophoresis

This paper reports a novel microfluidic-chip based platform using "phase-transfer magnetophoresis" enabling continuous biomolecule processing. As an example we demonstrate for the first time continuous DNA extraction from cell lysate on a microfluidic chip. After mixing bacterial Escherichia coli culture with superparamagnetic bead suspension, lysis and binding buffers, DNA is released from cells and captured by the beads. These DNA carrying beads are continuously transported across the interfaces between co-flowing laminar streams of sample mixture, washing and elution buffer. Bead actuation is achieved by applying a time-varying magnetic field generated by a rotating permanent magnet. Flagella-like chains of magnetic beads are formed and transported along the microfluidic channels by an interplay of fluid drag and periodic magnetic entrapment. The turnover time for DNA extraction was approximately 2 minutes with a sample flow rate of 0.75 μl s(-1) and an eluate flow rate of 0.35 μl s(-1). DNA recovery was 147% (on average) compared to bead based batch-wise extraction in reference tubes within a dilution series experiment over 7 orders of magnitude. The novel platform is suggested for automation of various magnetic bead based applications that require continuous sample processing, e.g. continuous DNA extraction for flow-through PCR, capture and analysis of cells and continuous immunoassays. Potential applications are seen in the field of biological safety monitoring, bioprocess control, environmental monitoring, or epidemiological studies such as monitoring the load of antibiotic resistant bacteria in waste water from hospitals.     

Removal of Cu2+ from aqueous solution by chitosan-coated magnetic nanoparticles modified with alpha-ketoglutaric acid

Chitosan-coated magnetic nanoparticles (CCMNPs), modified with a biodegradable and eco-friendly biologic reagent, alpha-ketoglutaric acid (alpha-KA), was used as a magnetic nanoadsorbent to remove toxic Cu(2+) ions from aqueous solution. The prepared magnetic nanoadsorbents were characterized by FTIR, TEM, VSM, XRD, and EDS. Factors influencing the adsorption of Cu(2+), e.g., initial metal concentration, initial pH, contact time and adsorbent concentration were investigated. TEM images show that the dimension of multidispersed circular particles is about 30 nm and no marked aggregation occurs. VSM patterns indicate superparamagnetic properties of magnetic nanoadsorbents. EDS pictures confirm the presence of the Cu(2+) on the surface of magnetic nanoadsorbents. Equilibrium studies show that Cu(2+) adsorption data follow Langmuir model. The maximum adsorption capacity (q(max)) for Cu(2+) ions was estimated to be 96.15 mg/g, which was higher than that of pure CCMNPs. The desorption data show no significant desorption hysteresis occurred. In addition, the high stability and recovery capacity of the chitosan-coated magnetic nanoparticles modified with alpha-ketoglutaric acid (alpha-KA-CCMNPs) suggest that these novel magnetic nanoadsorbents have potential applications for removing Cu(2+) from wastewater.     

Different methods for the fractionation of magnetic fluids

 Magnetic fluids are used in many fields of application, such as material separation and biomedicine. Magnetic fluids consist of magnetic nanoparticles, which commonly display a broad distribution of magnetic and nonmagnetic parameters. Therefore, upon application only a small number of particles contribute to the desired magnetic effect. In order to optimize magnetic fluids for applications preference is given to methods that separate magnetic nanoparticles according to their magnetic properties. Hence, a magnetic method was developed for the fractionation of magnetic fluids. Familiar size-exclusion chromatography of two different magnetic fluids was carried out for comparison. The fractions obtained and the original samples were also magnetically characterized by magnetic resonance and magnetorelaxometry, two biomedical applications. The size-exclusion fractions are similar to those of magnetic fractionation, despite the different separation mechanisms. In this respect, magnetic fractionation has several advantages in practical use over size-exclusion chromatography: the magnetic method is faster and has a higher capacity. The fractions obtained by both methods show distinctly different magnetic properties compared to the original samples and are therefore especially suited for applications such as magnetorelaxometry. Received: 12 July 1999/Accepted in revised form: 9 November 1999     

磁性纳米粒子在生物传感器中的应用研究进展

磁性纳米粒子是一种新型纳米材料,可应用于各种生物活性物质如蛋白质、DNA等的富集和分离,药物的磁靶向,以及疾病的诊断和治疗等许多领域。由于磁性纳米粒子有着独特的化学和物理性能,已经成功应用到磁控生物传感器、DNA传感器、蛋白质传感器、酶传感器以及其它类型的生物传感器中,并显著提高了生物传感器检测的灵敏度、缩短了生化反应的时间和提高检测的通量,为生物传感器领域开辟了广阔的应用前景。本文概述了磁性纳米粒子在生物传感器中的应用研究进展。     

Artificial cells in medicine and biotechnology

Since the feasibility of artificial cells was first demonstrated in 1957 [Chang (1, 2)], an increasing number of approaches to their preparation and use have become available. Thus artificial cell membranes can now be formed using a variety of synthetic or biological materials to produce desired variations in their permeability, surface properties, and blood compatibility. Almost any material can be included within artificial cells. These include enzyme systems, cell extracts, biological cells, magnetic materials, isotopes, antigens, antibodies, vaccines, hormones, adsorbents, and others. Since cells are the fundamental units of living organisms, it is not surprising that artificial cells can have a number of possible applications. This is especially so since artificial cells can be “tailor-made” to have very specialized functions. A number of potential applications suggested earlier have now reached a developmental stage appropriate for clinical trial or application. These clinical applications include the use of such cells as a red blood cell substitute, in hemoperfusion, in an artifical kidney or artificial liver, as detoxifiers, in an artificial pancreas, and so on. Artificial red blood cells based on lipid-coated fluorocarbon or crosslinked hemoglobin are being investigated in a number of centers. The principle of the artificial cells is also being used in biotechnology to immobilize enzymes and cells. Developments in biotechnology have also resulted in the use of the principle underlying the artificial cell to help produce interferons and monoclonal antibodies; to create immunosorbents; to develop an artificial pancreas; and to bring enzyme technology usefully into biotechnology and biomedical applications. Artificial cells are also being used as drug delivery systems based on slow release, on magnetic target delivery, on biodegradability, on liposomes, or other approaches. The present status and recent advances will be emphasized in this paper.