Nanomolar detection of hydrogen peroxide at a new polynuclear cluster of tin pentacyanonitrosylferrate nanoparticle-modified carbon ceramic electrode

This work describes a new electrochemical sensor for hydrogen peroxide based on tin pentacyanonitrosylferrate (SnPCNF)-modified carbon ceramic electrode (CCE). The modified electrode was constructed by using a sol-gel technique involving two steps: construction of CCE containing metallic tin (Sn) powder and then electrochemical creation of SnPCNF film on the surface of CCE. The modified electrode was characterized by energy-dispersive X-ray, Fourier transform infrared, scanning electron microscopy, and cyclic voltammetry (CV) techniques. The charge transfer coefficient (α) and charge transfer rate constant (k_s) for the modifying film were calculated. The electrocatalytic activity of the modified electrode toward the reduction of hydrogen peroxide was studied by CV and chronoamperometry. A linear calibration curve was obtained over the hydrogen peroxide concentration range of 0.5 to 69.4 μM using a hydrodynamic amperometric technique. The limit of detection (for a signal-to-noise ratio of 3) and sensitivity were found to be 92 nM and 0.89 μA/μM, respectively. Furthermore, the diffusion coefficient of hydrogen peroxide (D) and catalytic rate constant (k_cat) were calculated.     

Biosensors based on peptide exposure show single molecule conformations in live cells

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Characterization of proteins by means of their buffer capacity, measured with an ISFET-based coulometric sensor—actuator system

Proteins form the specific selector in many biochemical sensors. A change in one of the properties of such a protein has to be detected by an appropriate transducer, which completes the biochemical sensor. One of these properties is the buffer capacity of a protein. If the binding of a substance to a protein can significantly change the proton binding, which accounts for the buffer capacity of proteins, the detection of this changed buffer capacity enables the construction of a new type of biosensor.

It will be shown that the buffer capacity can be measured with an ISFET-based sensor—actuator device. The alternating generation of protons and hydroxyl ions by alternating current coulometry at a porous noble metal actuator electrode causes an associated small pH perturbation, which is detected by the underlying pH-sensitive ISFET. The amplitude of the measured signal is a function of the buffer capacity of the solute, in which proteins can be present (or these proteins can be adsorbed in the porous actuator electrode of the device). A model describing the transfer function from the electrical input signal of the actuator to the resulting chemical output, which is subsequently detected by the ISFET pH sensor, is presented. Preliminary results of the measured buffer capacity of ribonuclease and lysozyme are presented.     

Monitoring glutamine in animal cell cultures using a chemiluminescence fiber optic biosensor

Together with flow injection analysis (FIA), a chemiluminescence (CL) fiber optic biosensor system has been developed for determining glutamine in animal cell cultures. Glutaminase (GAH) and glutamate oxidase (GLO) were onto separate porous aminopropyl glass beads via glutaraldehyde activation and packed to form an enzyme column. These two enzymes acted in sequence on glutamine to produce hydrogen peroxide, which was then reacted with luminol in the presence of ferricyanide to produce a light signal. An anion exchanger was introduced on-line to eliminate interfering endogenous glutamate in view of its negative charge at pH above 3.22 (isoelectric pH). Among several resins tested, the acetate form was most effective, and this type of ion exchanger also effectively adsorbed uric acid, acetaminophen, and aspartic acid.There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 1 to 100 muM. A complete analysis could be performed in 2 min, including sampling and washing with a good reproducibility (+/- 4.4%). Both the bi-enzymic and ion exchange columns were useful for at least 500 analyses when the biosensor system was applied for the glutamine determination in murine hybridoma cell cultures and insect cell cultures. The values obtained compared well with those of HPLC, thus validating the applicability of the CL fiber optic system. (c) 1993 John Wiley & Sons, Inc.     

聚对苯二甲酸乙二醇酯水解酶检测方法的研究进展

聚对苯二甲酸乙二醇酯(polyethylene terephthalate,PET)是应用最广泛的合成聚酯之一。由于PET不易降解,在环境中积累,对陆地、水生生态系统以及人类健康构成严重威胁。基于生物酶催化的生物降解策略为PET回收利用提供了一种绿色途径,在过去20年间,已发现了多种PET水解酶,并通过蛋白质工程等手段来改善这些酶的降解性能,但是目前仍未找到适合大规模工业应用的PET水解酶。利用传统的检测方法筛选PET水解酶是一个缓慢而复杂的过程。为了促进PET酶法回收的工业化应用,需要研发高效的检测方法。近年来,研究人员开发了多种表征PET水解酶的分析方法。本文总结了可用于筛选PET水解酶的检测方法,如高效液相色谱法、紫外吸光度法和荧光激活液滴分选法等,并对其在筛选PET水解酶的应用方面进行了展望。     

应用FIA型微生物传感器测定谷氨酸含量的研究

目前应用酶或微生物细胞作为分子识别元件构建生物传感器测定谷氨酸含量的研究引起了广泛的兴趣,并陆续有实例报道。在这些报道中人们依据不同的酶催反应选择相应的离子选择性电极,如CO₂电极、NH₄⁺电极等,而测量对象有直接的测定谷氨酸,也有测定谷氨酸单钠的间接测定法。本文选用了可固定大量细胞的固定化细胞柱与流动注入法相结合的测量方法,并根据细胞柱的动力学模型分析动态响应曲线,计算测量结果,提高了测量精度,扩大了测量范围。     

Affinity immunosensor for milk progesterone: identification of critical parameters

The effects of several experimental parameters on the performance characteristics of a competitive-type immunochromatographic assay of milk progesterone were studied. Increasing the size of the colloidal gold particles used as a label increased both maximal signal obtained and sensitivity of the assay measured as slope of the progesterone standard curve. The concentration of the antibody used to prepare the detection zone was found to be a critical factor, in that low concentrations of antibody resulted in a poor sensitivity. The compatibilities of various buffer systems with the assay were studied. The assay worked well with buffers having a broad pH range of 4·5–8·5.     

An autoclavable glucose biosensor for microbial fermentation monitoring and control

The design, construction, and characterization of a prototype-regenerable glucose biosensor based on the reversible immobilization of glucose oxidase (GOx) using cellulose binding domain (CBD) technology is described. GOx, chemically linked to CBD, is immobilized by binding to a cellulose matrix on the sensor-indicating electode. Enzyme immobilization can be reversed by perfusing the cellulose matrix with a suitable eluting solution. An autocavable sensor membrane system is employed which is shown to be practical for use in real microbial fermentations. The prototype glucose biosensor was used without failure or deterioration during fed-batch fermentations of Escherichia coli reaching a maximum cell density of 85 g (dry weight)/L. Medium glucose concentration based on sensor output correlated closely with off-line glucose analysis and was controlled manually at 0.44 +/- 0.2 g/L for 2 h based on glucose sensor output. The sensor enzyme component could be eluted and replaced without interrupting the fermentation. To our knowledge, no other in situ biosensor has been used for such an extended period of time in such a high-cell-density fermentation. (c) 1995 John Wiley & Sons, Inc.     

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.     

A new biosensor for specific determination of sucrose using an oxidoreductase of Zymomonas mobilis and invertase

A new biosensor for specific determination of sucrose was developed using an oxidoreductase of Zymomonas mobilis and invertase. Cells of Z. mobilis were permeabilized with toluene in order to utilize the enzymes of glucose-fructose oxidoreductase and gluconolactonase inside the intact cells. Permeabilized cells and invertase were coimmobilized in a gelatin membrane, and a whole cell enzyme electrode was constructed by fixing the membrane on a pH electrode. The production of hydrogen ion was detected using the biosensor-connected microcomputer, and the concentration of sucrose was determined by using both the initial rate and the steady-state methods. Optimum conditions for biosensor response were pH 6.2 and temperature 35 degrees C. The effect of interfering compounds on the electrode response was investigated, and the interference by various sugars was eliminated by determining sucrose concentration using the steady-state method. The biosensor developed is simple and reproducible, and the calibration curve for sucrose is linear up to 70 g/L.