An amperometric biosensor based on horseradish peroxidase immobilized onto maize tassel-multi-walled carbon nanotubes modified glassy carbon electrode for determination of heavy metal ions in aqueous solution

A biosensor for trace metal ions based on horseradish peroxidase (HRP) immobilized on maize tassel-multiwalled carbon nanotube (MT-MWCNT) through electrostatic interactions is described herein. The biosensor was characterized using Fourier transform infrared (FTIR), UV–vis spectrometry, voltammetric and amperometric methods. The FTIR and UV–vis results inferred that HRP was not denatured during its immobilization on MT-MWCNT composite. The biosensing principle was based on the determination of the cathodic responses of the immobilized HRP to H₂O₂, before and after incubation in trace metal standard solutions. Under optimum conditions, the inhibition rates of trace metals were proportional to their concentrations in the range of 0.092–0.55 mg L⁻¹, 0.068–2 mg L⁻¹ for Pb²⁺ and Cu²⁺ respectively. The limits of detection were 2.5 μg L⁻¹ for Pb²⁺ and 4.2 μg L⁻¹ for Cu²⁺. Representative Dixon and Cornish-Bowden plots were used to deduce the mode of inhibition induced by the trace metal ions. The inhibition was reversible and mixed for both metal ions. Furthermore, the biosensor showed good stability, selectivity, repeatability and reproducibility.     

Piezoelectric urea biosensor based on immobilization of urease onto nanoporous alumina membranes

The urease was immobilized onto nanoporous alumina membranes prepared by the two-step anodization method, and a novel piezoelectric urea sensing system with separated porous alumina/urease electrode has been developed through measuring the conductivity change of immobilized urease/urea reaction. The process of urease immobilization was optimized and the performance of the developed urea biosensor was evaluated. The obtained urea biosensor presented high-selectivity monitoring of urea, better reproducibility (S.D. = 0.02, n = 6), shorter response time (30 s), wider linear range (0.5 μM to 3 mM), lower detection limit (0.2 μM) and good long-term storage stability (with about 76% of the enzymatic activity retained after 30 days). The clinical analysis of the urea biosensor confirmed the feasibility of urea detection in urine samples.     

Chemiluminescence flow-through biosensor for glucose with eggshell membrane as enzyme immobilization platform

In this study, a new chemiluminescence (CL) flow-through biosensor for glucose was developed by immobilizing glucose oxidase (GOD) and horseradish peroxidase (HRP) on the eggshell membrane with glutaraldehyde as a cross-linker. The CL detection involved enzymatic oxidation of glucose to D-gluconic acid and hydrogen peroxide (H2O2) and then H2O2 oxidizing luminol to produce CL emission in the presence of HRP. The immobilization condition (e.g., immobilization time, GOD/HRP ratio, glutaraldehyde concentration) was studied in detail. It showed good storage stability at 4 degrees C over a 5-month period. The proposed biosensor exhibited short response time, high sensitivity, easy operation, and simple sensor assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum.     

Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.     

Covalent immobilization of glucose oxidase onto poly(styrene-co-glycidyl methacrylate) monodisperse fluorescent microspheres synthesized by dispersion polymerization

In this study, micron-sized poly(styrene-co-glycidyl methacrylate) (PSt-GMA) fluorescent microspheres of 5.1microm in diameter were synthesized via dispersion polymerization of styrene and glycidyl methacrylate in the presence of 1,4-bis(5-phenyloxazol-2-yl) benzene (POPOP), which provided surface functional groups for covalent immobilization of enzymes. In an effort to study the biocompatibility of the microspheres' surface, glucose oxidase and beta-d-(+)-glucose were selected as a catalytic system for enzymatic assays. A colorimetric method was adopted in evaluating enzymatic activity by introducing horseradish peroxidase (HRP). Both the immobilization amount and the apparent activity of immobilized glucose oxidase from Aspergillus niger (GOD) were determined at different conditions. The results show that the immobilized enzymes retained approximately 28 to 34% activity, as compared with free enzymes, without pronounced alteration of the optimum pH and temperature. Kinetics studies show that the corresponding values of K(m) and V(max) are 23.2944 mM and 21.6450M/min.mg GOD for free enzymes and 35.1780 mM and 15.4799M/min.mg GOD for immobilized enzymes. The operational stability studies show that immobilized GOD could retain nearly 50% initial activity after being washed 20 times. The results suggest that the resultant PSt-GMA fluorescent microspheres provide a suitable surface for covalent immobilizing biomolecules; therefore, they have the potential of being used in fluorescence-based immunoassays in high-throughput screening or biosensors.     

基于酶生物传感器法检测果蔬中毒死蜱残留的提取工艺研究

使用自主研制的酶生物传感器型农残检测仪进行农药残留检测,通过筛选可测果蔬种类、调整优化样品处理大小、样品加标后静置时间、样品与提取液比例和振荡提取速度,提高检测用酶对农药的抑制敏感性,从而达到降低农药检出限、提高回收率的目的。主要设置的参数如下:样品处理方式分为打碎、切碎(切成1 cm×1 cm和1. 5 cm×3 cm大小);加标后静置时间为5 min、15min、30 min、60 min和90 min;料液比为1∶1、1∶2. 5、1∶5、1∶7. 5和1∶10;振荡提取速度为0 r/min、110 r/min、190 r/min和225 r/min;主要测定的农药为毒死蜱。结果发现,不同果蔬品种对固定化酶的抑制率影响小,样品大小为1 cm×1 cm、加标后静置时间为5 min、料液比为1∶1、加入提取剂后的振荡速度为190 r/min为最佳前处理方式组合。酶生物传感器农残检测仪能够满足快速检测果蔬中有机磷农药残留的需要。     

Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.     

Recent trends using natural polymeric nanofibers as supports for enzyme immobilization and catalysis

All the disciplines of science, especially biotechnology, have given continuous attention to the area of enzyme immobilization. However, the structural support made by material science intervention determines the performance of immobilized enzymes. Studies have proven that nanostructured supports can maintain better catalytic performance and improve immobilization efficiency. The recent trends in the application of nanofibers using natural polymers for enzyme immobilization have been addressed in this review article. A comprehensive survey about the immobilization strategies and their characteristics are highlighted. The natural polymers, e.g., chitin, chitosan, silk fibroin, gelatin, cellulose, and their blends with other synthetic polymers capable of immobilizing enzymes in their 1D nanofibrous form, are discussed. The multiple applications of enzymes immobilized on nanofibers in biocatalysis, biosensors, biofuels, antifouling, regenerative medicine, biomolecule degradation, etc.; some of these are discussed in this review article.     

An amperometric enzyme biosensor for real‐time measurements of cellobiohydrolase activity on insoluble cellulose

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi‐crystalline and amorphous, can be monitored directly and in real‐time by an enzyme‐modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross‐linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH‐catalyzed reaction with cellobiose, was recorded under constant‐potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH‐biosensors showed high sensitivity (87.7 µA mM^?1 cm^?2), low detection limit (25 nM), and fast response time (t_95% ～ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH‐biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the β‐anomer of cello‐oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real‐time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 2012; 109: 3199–3204. © 2012 Wiley Periodicals, Inc.     

Purification of horse radish peroxidase by metal affinity chromatography in an expanded bed system

Immobilised metal chelate affinity chromatography (IMAC) in an expanded bed mode was used for the purification of horse radish peroxidase. Recovery of horse radish peroxidase varied between 85 and 72% starting from the crude homogenate. When a pure peroxidase was passed through the purification protocol a recovery of about 95% was achieved.     

Amperometric hydrogen peroxide biosensor based on the immobilization of HRP on nano-Au/Thi/poly (p-aminobenzene sulfonic acid)-modified glassy carbon electrode

A novel hydrogen peroxide biosensor was fabricated for the determination of H(2)O(2). The precursor film was first electropolymerized on the glassy carbon electrode with p-aminobenzene sulfonic acid (p-ABSA) by cyclic voltammetry (CV). Then thionine (Thi) was adsorbed to the film to form a composite membrane, which yielded an interface containing amine groups to assemble gold nanoparticles (nano-Au) layer for immobilization of horseradish peroxidase (HRP). The electrochemical characteristics of the biosensor were studied by CV and chronoamperometry. The factors influencing the performance of the resulting biosensor were studied in detail. The biosensor responded to H(2)O(2) in the linear range from 2.6 x 10(-6) mol/L to 8.8 x 10(-3) mol/L with a detection limit of 6.4 x 10(-7) mol/L. Moreover, the studied biosensor exhibited good accuracy and high sensitivity. The proposed method was economical and efficient, making it potentially attractive for the application to real sample analysis.     

酶的固定化技术最新研究进展

酶是一种高效、绿色、应用广泛的生物催化剂,因其固定化形态在多种性质上均优于游离态,酶固定化技术应运而生并不断发展。我国固定化技术研究始于20世纪70年代,目前固定化酶在食品、医疗、能源、环境治理等领域得到了广泛的应用,但现有固定化技术仍存在适用范围小、成本较高等缺陷。因此,在较为成熟的传统固定化技术基础上,研究者们对新型固定化技术的研究与创新进行了大量尝试,形成了一批以固定化载体和固定化方式为核心的新型固定化技术。文中作者结合团队十余年对固定化技术的研究和理解,归纳介绍了新型酶固定化技术的发展方向和应用趋势,并阐述了对固定化技术未来发展的理解和建议。     

Disposable biosensor based on enzyme immobilized on Au-chitosan-modified indium tin oxide electrode with flow injection amperometric analysis

Indium tin oxide (ITO) electrode is used to fabricate a novel disposable biosensor combined with flow injection analysis for the rapid determination of H2O2. The biosensor is prepared by entrapping horseradish peroxidase (HRP) enzyme in colloidal gold nanoparticle-modified chitosan membrane (Au-chitosan) to modify the ITO electrode. The biosensor is characterized by scanning electron microscope, atomic force microscope, and electrochemical methods. Parameters affecting the performance of the biosensor, including concentrations of o-phenylenediamine (OPD) and pH of substrate solution, were optimized. Under the optimal experimental conditions, H2O2 could be determined in the linear calibration range from 0.01 to 0.5 mM with a correlation coefficient of 0.997 (n=8). The amperometric response of the biosensor did not show an obvious decrease after the substrates were injected continuously 34 times into the flow cell. The prepared biosensor not only is economic and disposable, due to the low-cost ITO film electrode obtained from industrial mass production, but also is capable with good detection precision, acceptable accuracy, and storage stability for the fabrication in batch.     

Covalent immobilization of redox enzyme on electrospun nonwoven poly(acrylonitrile-co-acrylic acid) nanofiber mesh filled with carbon nanotubes: a comprehensive study

In this work, novel conductive composite nanofiber mesh possessing reactive groups was electrospun from solutions containing poly(acrylonitrile-co-acrylic acid) (PANCAA) and multi-walled carbon nanotubes (MWCNTs) for redoxase immobilization, assuming that the incorporated MWCNTs could behave as electrons transferor during enzyme catalysis. The covalent immobilization of catalase from bovine liver on the neat PANCAA nanofiber mesh or the composite one was processed in the presence of EDC/NHS. Results indicated that both the amount and activity retention of bound catalase on the composite nanofiber mesh were higher than those on the neat PANCAA nanofiber mesh, and the activity increased up to 42%. Kinetic parameters, K(m) and V(max), for the catalases immobilized on the composite nanofiber mesh were lower and higher than those on the neat one, respectively. This enhanced activity might be ascribed to either promoted electron transfer through charge-transfer complexes and the pi system of carbon nanotubes or rendered biocompatibility by modified MWCNTs. Furthermore, the immobilized catalases revealed much more stability after MWCNTs were incorporated into the polymer nanofiber mesh. However, there was no significant difference in optimum pH value and temperature, thermal stability and operational stability between these two immobilized preparations, while the two ones appeared more advantageous than the free in these properties. The effect of MWCNTs incorporation on another redox enzyme, peroxidase, was also studied and it was found that the activity increased by 68% in comparison of composite one with neat preparation.     

Amperometric biosensor based on coentrapment of enzyme and mediator by gold nanoparticles on indium-tin oxide electrode

A disposable pseudo-mediatorless amperometric biosensor has been fabricated for the determination of hydrogen peroxide (H2O2). In the current study, an indium-tin oxide (ITO) electrode was modified with thiol functional group by (3-mercaptopropyl)trimethoxysilane. The stable nano-Au-SH monolayer (AuS) was then prepared through covalent linking of gold nanoparticles and thiol groups on the surface of the ITO. The horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) were finally coentrapped by the colloidal gold nanoparticles. The immobilized TMB was used as an electron transfer mediator that displayed a surface-controlled electrode process at a scan rate of less than 50mV/s. The biosensor was characterized by photometric and electrochemical measurements. The results showed that the prepared AuS monolayer not only could steadily immobilize HRP but also could efficiently retain HRP bioactivity. Parameters affecting the performance of the biosensor, including the concentrations of the immobilized TMB and HRP, the pH value, and the reaction temperature, were optimized. Under the optimized experimental conditions, H(2)O(2) could be determined in a linear calibration range from 0.005 to 1.5mM with a correlation coefficient of 0.998 (n=14) and a detection limit of 1microM at a signal/noise ratio of 3. The proposed method provides a new alternative to develop low-cost biosensors by using ITO film electrodes from industrial mass production.     

Polyphenol biosensor based on laccase immobilized onto silver nanoparticles/multiwalled carbon nanotube/polyaniline gold electrode

Laccase purified from Ganoderma sp. was immobilized covalently onto electrochemically deposited silver nanoparticles (AgNPs)/carboxylated multiwalled carbon nanotubes (cMWCNT)/polyaniline (PANI) layer on the surface of gold (Au) electrode. A polyphenol biosensor was fabricated using this enzyme electrode (laccase/AgNPs/cMWCNT/PANI/Au electrode) as the working electrode, Ag/AgCl as the reference electrode, and platinum (Pt) wire as the auxiliary electrode connected through a potentiostat. The biosensor showed optimal response at pH 5.5 (0.1 M acetate buffer) and 35 °C when operated at a scan rate of 50 mV s⁻¹. Linear range, response time, and detection limit were 0.1–500 μM, 6 s, and 0.1 μM, respectively. The sensor was employed for the determination of total phenolic content in tea, alcoholic beverages, and pharmaceutical formulations. The enzyme electrode was used 200 times over a period of 4 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors in that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective PANI microenvironment.     

A two‐enzyme immobilization approach using carbon nanotubes/silica as support

Multiple enzyme mixtures are attractive for the production of many compounds at an industrial level. We report a practical and novel approach for coimmobilization of two enzymes. The system consists of a silica microsphere core coated with two layers of individually immobilized enzymes. The model enzymes α‐amylase (AA) and glucoamylase (GluA) were individually immobilized on carbon nanotubes (CNTs). A CNT‐GluA layer was formed by adsorbing CNT‐GluA onto silica microsphere. A sol‐gel layer with entrapped CNT‐AA was then formed outside the CNT‐GluA/silica microsphere conjugate. The coimmobilized α‐amylase and glucoamylase exhibited 95.1% of the activity of the mixture of free α‐amylase and glucoamylase. The consecutive use exhibited a good stability of the coimmobilized enzymes. The developed approach demonstrates advantages, including controlling the ratio of coimmobilized enzymes in an easy way, facilitating diffusion of small molecules in and out of the matrix, and preventing the leaching of enzymes. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:42–47, 2015     

Electrochemically platinized carbon paste enzyme electrodes: A new design of amperometric glucose biosensors

A platinized carbon paste prepared via electrodeposition had a preferential electrocatalytic action toward H₂O₂. Therefore, we have developed a new amperometric glucose biosensor based on the immobilization of glucose oxidase on to the electrochemically platinized carbon paste. The proposed biosensor is free of potential interferences due to its cathodic detection of glucose at the potential of 0.0 V (vs. Ag/AgCl). It also shows acceptable analytical performance in terms of linearity (6 × 10⁻⁵ to 1.2 × 10⁻² M, r = 0.998), detection limit (2 × 10⁻⁵ M), response time (20–30 s), reproducibility (RSD = 4.4%), and storage life (t _0.80 = 45 days). All these advantages of the biosensor raise potential possibilities for its medical or other biotechnical applications.     

Retention of enzyme by electropolymerized film: a new approach in developing a hypoxanthine biosensor

An amperometric biosensor for hypoxanthine was constructed by forming a layer of crosslinked xanthine oxidase on a platinum electrode, followed by electropolymerization of a submonolayer film of resorcinol and para-diaminobenzene. The fabricated electrodes were evaluated for speed of response, sensitivity, and reusability. Optimal performance was obtained with enzyme-based electrodes sparsely covered with film which was formed by electropolymerization in less than 6 min. The resulting electrodes exhibited linear response to hypoxanthine in the. range 5-300 muM with a response time of 2 min. Application of the biosensor in monitoring hypoxanthine content of fish extracts yielded results which agreed well with spectrophotometric assays using soluble xanthine oxidase. The biosensor was stable for 60 days when stored at 4 degrees C in phosphate buffer and it could be used continuously for 6 h with over 50 assays.