共查询到20条相似文献,搜索用时 15 毫秒
1.
Whole‐cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production
下载免费PDF全文
![点击此处可从《Biotechnology and bioengineering》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Lisa Goers Catherine Ainsworth Cher Hui Goey Cleo Kontoravdi Paul S. Freemont Karen M. Polizzi 《Biotechnology and bioengineering》2017,114(6):1290-1300
Many high‐value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole‐cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole‐cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole‐cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole‐cell biosensors. Biotechnol. Bioeng. 2017;114: 1290–1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. 相似文献
2.
In this paper, a mediatorless amperometric glucose biosensor based on direct covalent immobilisation of monomolecular layer of glucose oxidase (GOx) on a semiconducting indium-tin oxide (ITO) is demonstrated. The abundance of surface hydroxyl functional group of ITO allows it to be used as a suitable platform for direct covalent immobilisation of the enzyme for sensor architecture. The anodic current corresponding to electrochemical oxidation of the enzymatic product, hydrogen peroxide, at a sputtered Pt electrode at 0.500 V (vs. SCE) was obtained as the sensor signal. It was found that the biosensor based on the direct immobilisation scheme shows a fast biosensor response, minimum interference from other common metabolic species and ease of biosensor miniaturisation. A linear range of 0-10 mM of glucose was demonstrated, which exhibits a high sensitivity as far as performance per immobilised GOx molecule is concerned. A detection limit as low as 0.05 mM and long-term stability were observed. Even more important, the biosensor design allows fabrication through a dry process. These characteristics make it possible to achieve mass production of biosensor compatible with the current electronic integrated circuit manufacturing technologies. 相似文献
3.
A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity. 相似文献
4.
全细胞生物传感器是一种以微生物全细胞为敏感元件,可以快速感应环境中的毒性物质及污染物的装置。因其具有响应快、体积小、成本低、可实现原位监测等优势,在环境监测、药品研发、食品工业等领域显示出巨大潜力。综述了全细胞生物传感器的原理、分类及其在环境污染物监测领域的应用进展等,并对其未来的发展趋势进行了展望,以期为全细胞生物传感器的开发与利用提供参考。 相似文献
5.
The in vitro metabolic stability testing on synthetic obestatin peptides from two different species (human hOb and mouse mOb) using HPLC analysis is described. A reversed-phase C(18) column of 300A pore size was used, with a gradient system based on aqueous formic acid and acetonitrile. Electrospray ionization (ESI) ion trap mass spectrometry was used for identification of the chromatographic eluting peptide metabolic products, while UV (DAD) and fluorescence served quantitative purposes. Differences in the metabolic degradation kinetics of hOb and mOb were found in plasma, liver and kidney homogenate, with half-lives ranging between 12.6 and 138.0min. Proteolytic hydrolysis at the N-terminal Phe residue and cleavage at Pro(4)-Phe(5) were found to be two major metabolic pathways, accounting for more than 50% of the metabolic degradation. Several other labile peptide bonds were located. The influence of a standard protease inhibitor cocktail was investigated, as well as the metabolism of iodinated human obestatin in liver homogenate. Our results indicate that the major instability of obestatin peptides, as currently used in biomedical investigations, should be taken into account in the interpretation of the obtained results. 相似文献
6.
A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity. 相似文献
7.
A microbial biosensor, using Acetobacter pasteurianus cells and an oxygen electrode, was developed for the determination of lactic acid. The bacterial cells were retained on a nylon membrane and attached to the surface of the oxygen electrode. In view of response time, stability and sensitivity, the biosensor performed best at 26°C and in pH 6 phthalate buffer containing magnesium sulfate. The activity of the retained cells was stable for approximately 170 h and was regenerable. The biosensor exhibited a hyperbolic response to both D- and L-lactic acid in the range of 10−4 M to 25 × 10−3 M. However, in the range 10−4 M to 15 × 10−4 M the response was linear. The microbial biosensor was applicable for detecting lactate concentration in yogurt and milk, since it was not sensitive to lactose, sucrose and glucose — three major components of such dairy products. 相似文献
8.
Single-use conductivity and microbial sensors were used to investigate the effect of both species (chloride, nitrate, and sulphate) and concentration/osmolarity of anions on the metabolic activity of Escherichia coli. A new disposable, single-use conductivity sensor is described which is compatible with the CellSense mediated amperometric biosensor system. The effect of changing salinity and nitrate concentration on the response of E. coli to 3,5-dichlorophenol and mercuric chloride was determined. The implications for toxicity assessment of a hybrid sensing system, allowing the simultaneous monitoring of physico-chemical and biological data, are discussed. 相似文献
9.
In this study, an amperometric carbon paste biosensor is developed for glucose-6-phosphate (G6P) monitoring which is based on entrapped Mg2+ ions, G6P dehydrogenase, NADP+ polyethylenimine (PEI) and the electroactive mediator, tetracyanoquinodimethane (TCNQ). The calibration line had a slope of 1.55 x 10(-5) A. M-1 with a correlation coefficient of 0.9965. The limit of detection (defined as three times the standard deviation of the response of the electrode to blank phosphate buffer injections (noise)) of the G6P biosensor was 5.0 x 10(-5) M. The application of this biosensor for monitoring G6P in human blood using the standard addition method is also demonstrated. A two-parameter empirical equation which adequately describes the deactivation of the biosensor steady-state response with time is also proposed. 相似文献
10.
4-羟基苯甲酸(4HBA)是在自然界中广泛存在的芳香族化合物,也是很多天然产物和人工合成化合物的中间代谢产物。4HBA的代谢途径有原儿茶酸开环途径、脱碳酸途径和厌氧微生物的苯甲酰-CoA还原途径,以及尚未完全阐明的龙胆酸开环途径。从4HBA转化为龙胆酸的过程包含NIH重排反应步骤,本综述重点介绍NIH重排反应的研究进展并初步介绍了涉及4HBA降解过程中的酶。在本综述中,结合我们的研究工作介绍了一个嗜热Bacillus sp.B1菌株降解4HBA等芳香族化合物的代谢途径,最后对4HBA降解过程中的NIH重排反应研究进行了展望。 相似文献
11.
A thick-film phosphate biosensor based on hydrogel immobilized pyruvate oxidase (POD) has been developed for rapid phosphate process control monitoring in an experimental sequencing batch reactor (SBR) system. We have employed a phosphate biosensor in an off-line monitoring of phosphate concentrations in a bench scale SBR. Measurements with biosensor show a good correlation (r2=0.98) with those of commercial colorimetric phosphate testing kits. The signal response time was 1 min with a detection limit of 5 microM. The biosensor method showed a good operational stability, needed less experimental procedures and a small sample size (approximately 20 microl). This allows its practical application for rapid phosphate measurements to obtain real time process data in a SBR system. 相似文献
12.
A biosensor based on pyruvate oxidase (POX) enzyme was developed for the investigation of the effect of thiamine (vitamin B(1)) molecule on the activity of the enzyme. The biosensor was prepared with a chemical covalent immobilization method on the dissolved oxygen (DO) probe by using gelatin and cross-linking agent, glutaraldehyde. POX catalyzes the degradation of pyruvate to acetylphosphate, CO(2) and H(2)O(2) in the presence of phosphate and oxygen. Thiamine is an activator for POX enzyme and determination method of the biosensor was based on this effect of thiamine on the activity of the enzyme. The biosensor responses showed increases in the presence of thiamine. Increases in the biosensor responses were related to thiamine concentration. Thiamine determination is based on the assay of the differences on the biosensor responses on the oxygenmeter in the absence and the presence of thiamine. The biosensor response depend linearly on thiamine concentration between 0.025 and 0.5 microM with 2 min response time. In the optimization studies of the biosensor the most suitable enzyme amount was found as 2.5 U cm(-2) and also phosphate buffer (pH 7.0; 50 mM) and 35 degrees C were obtained as the optimum working conditions. In the characterization studies of the biosensor some parameters such as activator and interference effects of some substances on the biosensor response and reproducibility were carried out. 相似文献
13.
An amperometric biosensor based on a composite of single-walled carbon nanotubes, plasma-polymerized thin film, and an enzyme 总被引:1,自引:0,他引:1
We report on an amperometric biosensor that is based on a nanocomposite of carbon nanotubes (CNT), a nano-thin plasma-polymerized film (PPF), and glucose oxidase (GOx) as an enzyme model. A mixture of the GOx and a CNT film is sandwiched with 10-nm-thick acetonitrile PPFs. Under PPF layer was deposited onto a sputtered gold electrode. To facilitate the electrochemical communication between the CNT layer and GOx, CNT was treated with nitrogen or oxygen plasma. The resulting device showed that the oxidizing current response due to enzymatic reaction was 4-16-fold larger than that with only CNT or PPF, showing that the PPF and/or plasma process is an enzyme-friendly platform for designing electrochemical communication from the reaction center of GOx to the electrode via CNTs. The optimized glucose biosensor showed high sensitivity (sensitivity of 42 microA mM(-1)cm(-2), correlation coefficient of 0.992, linear response range of 0.025-2.2 mM, and a detection limit of 6 microM at signal/noise ratio of 3, +0.8 V versus Ag/AgCl), high selectivity (almost no interference by 0.5 mM ascorbic acid) for glucose quantification, and rapid response (<4 s to reach 95% of maximum response). Additionally, the devices showed a small and stable background current (0.35+/-0.013 microA) compared with the glucose response (ca. 10 microA at 10mM glucose) and suitable reproducibility from sample-to-sample (<3%, n=4). 相似文献
14.
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. 相似文献
15.
Optical Waveguide Lightmode Spectroscopy (OWLS) is based on measurements of the effective refractive index of a thin layer above the waveguide. Its potential as a whole-cell biosensor was demonstrated recently monitoring adhesion and spreading of Baby Hamster Kidney (BHK) cells on-line. In this work the OWLS is shown to be a promising tool to study the adhesion, morphology and metabolic state of fibroblasts in real time. A new design of the measuring chamber allowed simultaneous observation by phase-contrast microscopy and made the adsorbed cell density controlable and reproducible. The OWLS signal correlated quantitatively with the contact-area between the fibroblasts and the waveguide. The OWLS signals for adhesion and spreading of three different fibroblast cell lines were in good agreement with their morphology identified by phase-contrast microscopy. The cell adhesion and cell shape changes were examined in three scenarios: (a) serum-induced spreading of the surface attached fibroblasts was followed until it was completed, and the OWLS signal remained constant for over 12 h; (b) the fully spread cells were exposed to the microtubuli-disrupting colchicine and a decrease of the OWLS signal was monitored; (c) in a similar experiment with benzalkonium chloride, a strong skin irritant, a concentration-dependent response of the signal was found. The results show the strength of the OWLS method for monitoring the adhesion behavior of anchorage-dependent cells such as fibroblasts. It has a great potential as a whole-cell biosensor for high throughput screening in toxicology. 相似文献
16.
Due to their frequent occurrence in food, cosmetics and pharmaceutical products, and their poor solubility in water, the detection of peroxides in organic solvents has aroused significant interest. For diagnostics or on-site testing, a fast and specific experimental approach is required. Although aqueous peroxide biosensors are well known, they are usually not suitable for nonaqueous applications due to their instability. Here we describe an organic phase biosensor for hydrogen peroxide based on horseradish peroxidase immobilized in an Eastman AQ 55 polymer matrix. Rotating disc amperometry was used to examine the effect of the solvent properties, the amount and pH of added buffer, the concentration of peroxide and ferrocene dimethanol, and the amount of Eastman AQ 55 and of enzyme on the response of the biosensor to hydrogen peroxide. The response of the biosensor was limited by diffusion. Linear responses (with detection limits to hydrogen peroxide given in parentheses) were obtained in methanol (1.2 microM), ethanol (0.6 microM), 1-propanol (2.8 microM), acetone (1.4 microM), acetonitrile (2.6 microM), and ethylene glycol (13.6 microM). The rate of diffusion of ferrocene dimethanol was more constrained than the rate of diffusion of hydrogen peroxide, resulting in a comparatively narrow linear range. The main advantages of the sensor are its ease of use and a high degree of reproducibility, together with good operational and storage stability. 相似文献
17.
A new amperometric whole cell biosensor based on Saccharomyces cerevisiae immobilized in gelatin was developed for selective determination of vitamin B1 (thiamine). The biosensor was constructed by using gelatin and crosslinking agent glutaraldehyde to immobilize S. cerevisiae cells on the Teflon membrane of dissolved oxygen (DO) probe used as the basic electrode system combined with a digital oxygen meter. The cells were induced by vitamin B1 in the culture medium, and the cells used it as a carbon source in the absence of glucose. So, when the vitamin B1 solution is injected into the whole cell biosensor system, an increase in respiration activity of the cells results from the metabolic activity and causes a decrease in the DO concentration of interval surface of DO probe related to vitamin B1 concentration. The response time of the biosensor is 3 min, and the optimal working conditions of the biosensor were carried out as pH 7.0, 50mM Tris-HCl, and 30 degrees C. A linear relationship was obtained between the DO concentration decrease and vitamin B1 concentration between 5.0 x 10(-3) and 10(-1) microM. In the application studies of the biosensor, sensitive determination of vitamin B1 in the vitamin tablets was investigated. 相似文献
18.
Zhong Z Fritzsche M Pieper SB Wood TK Lear KL Dandy DS Reardon KF 《Biosensors & bioelectronics》2011,26(5):2407-2412
Measurements of pollutants such as toluene are critical for the characterization of contaminated sites and for the monitoring of remediation processes and wastewater treatment effluents. Fiber optic enzymatic biosensors have the potential to provide cost-effective, real time, continuous, in situ measurements. In this study, a fiber optic enzymatic biosensor was constructed and characterized for the measurement of toluene concentrations in aqueous solutions. The biological recognition element was toluene ortho-monooxygenase (TOM), expressed by Escherichia coli TG1 carrying pBS(Kan)TOM, while an optical fiber coated with an oxygen-sensitive ruthenium-based phosphorescent dye served as the transducer. Toluene was detected based on the enzymatic reaction catalyzed by TOM, which resulted in the consumption of oxygen and changes in the phosphorescence intensity. The biosensor was found to have a limit of detection of 3 μM, a linear signal range up to 100 μM, and a response time of 1 h. The performance was reproducible with different biosensors (RSD=7.4%, n=8). The biosensor activity declined with each measurement and with storage time, particularly at elevated temperatures. This activity loss could be partially reversed by exposure to formate, suggesting that NADH consumption was the primary factor limiting lifetime. This is the first report of an enzymatic toluene sensor and of an oxygenase-based biosensor. Since many oxygenases have been reported, the design concept of this oxygenase-based biosensor has the potential to broaden biosensor applications in environmental monitoring. 相似文献
19.
A previously developed biosensor for fast estimation of short-term biochemical oxygen demand (BODst) was used for off-line monitoring of intermediate products from the initial step of an anaerobic process in laboratory scale. Good agreement was generally achieved between the results from the biosensor method and the conventional 5-day test except for samples with high content of organic polymers. During the period of agreement between the measurement principles, good correlation was achieved between the biogas production rate and the organic loading rate. The results from this study demonstrate that BODst can be a successful monitoring parameter to achieve a better process control. 相似文献
20.
A novel piezoelectric sensor has been developed for bilirubin (BR) detection, based on the modification of molecularly imprinted hydroxyapatite (HAP) film onto a quartz crystal by molecular imprinting and surface sol-gel technique. The performance of the developed BR biosensor was evaluated and the results indicated that a sensitive BR biosensor could be fabricated. The obtained BR biosensor presents high-selectivity monitoring of BR, better reproducibility, shorter response time (37 min), wider linear range (0.05-80μM) and lower detection limit (0.01μM). The analytical application of the BR biosensor confirms the feasibility of BR detection in serum sample. 相似文献