Development of a fully integrated microdistillation flow injection system for the determination of trace level ammonia

An in-housed designed computerised flow injection system comprised a fully integrated microdistillation flow injection (MDFI) system for low level ammonia analysis was reported. In this system, the microdistillation separation step was incorporated into the flow injection manifold and the ammonia gas sensing probe sensing element was replaced by a flow-through micro-pH electrode which sensed the change in pH of a flowing collector solution caused by the dissolution of distilled ammonia gas, in a process analogous to that occurring in the internal solution of the gas sensing probe. A computerised control and data acquisition system was constructed for this system using a commercially available data acquisition card which offered many advantages such as improved data acquisition rates and control over the system components, as well as good graphics display and data processing options. The system was optimised using a multi-variable simplex optimisation technique.     

An intelligent sensor system for the determination of ammonia using flow injection analysis

An intelligent automated ammonia monitoring system was developed based on a commercial ammonia selective electrode used in flow injection analysis (FIA) mode. A prototype automatic monitoring system was produced and interfaced to an IBM personal computer. The interfacing involved the design and fabrication of a sensor interface, an inter-integrated circuit (I²C) card and a flow injection analysis controller. This ammonia monitoring system will be used in conjunction with the dissolved oxygen and temperature sensors for the determination of ammonia toxicity. Use of a sodium hydroxide reagent line allowed determination of total ammonia (un-ionized (NH₃) + ionized (NH₄⁺)). With the output of the pH and temperature probes, un-ionized ammonia can be determined based on an equilibrium calculation. This experimental system was controlled under an expert system, Crystal. It provides the knowledge of equipment setup, control and results interpretation based on the rules stored in its knowledge base.     

A flow injection flow cytometry system for on-line monitoring of bioreactors

For direct and on-line study of the physiological states of cell cultures, a robust flow injection system has been designed and interfaced with flow cytometry (FI-FCM). The core of the flow injection system includes a microchamber designed for sample processing. The design of this microchamber allows not only an accurate on-line dilution but also on-line cell fixation, staining, and washing. The flow injection part of the system was tested by monitoring the optical density of a growing E.coli culture on-line using a spectrophotometer. The entire growth curve, from lag phase to stationary phase, was obtained with frequent sampling. The performance of the entire FI-FCM system is demonstrated in three applications. The first is the monitoring of green fluorescent protein fluorophore formation kinetics in E.coli by visualizing the fluorescence evolution after protein synthesis is inhibited. The data revealed a subpopulation of cells that do not become fluorescent. In addition, the data show that single-cell fluorescence is distributed over a wide range and that the fluorescent population contains cells that are capable of reaching significantly higher expression levels than that indicated by the population average. The second application is the detailed flow cytometric evaluation of the batch growth dynamics of E.coli expressing Gfp. The collected single-cell data visualize the batch growth phases and it is shown that a state of balanced growth is never reached by the culture. The third application is the determination of distribution of DNA content of a S. cerevisiae population by automatically staining cells using a DNA-specific stain. Reproducibility of the on-line staining reaction shows that the system is not restricted to measuring the native properties of cells; rather, a wider range of cellular components could be monitored after appropriate sample processing. The system is thus particularly useful because it operates automatically without direct operator supervision for extended time periods.     

On-line heat flux measurements improve the culture medium for the growth and productivity of genetically engineered CHO cells

With the increasingly competitive commercial production of target proteins by hybridoma and genetically engineered cells, there is an urgent requirement for biosensors to monitor and control on-line and in real time the growth of cultured cells. Since growth is accompanied by an enthalpy change, heat dissipation measured by calorimetry could act as an index for metabolic flow rate. Recombinant CHO cell suspensions producing interferon-γ were pumped to an on-line flow calorimeter. The results showed that an early reflection of metabolic change is size-specific heat flux obtained from dividing heat flow rate by the capacitance change of the cell suspension, using the on-line probe of a dielectric spectroscope. Comparison of heat flux with glucose and glutamine fluxes indicated that the former most accurately reflected decreased metabolic activity. Possibly this was due to accumulation of lactate and ammonia resulting from catabolic substrates being used as biosynthetic precursors. Thus, the heat flux probe is an ideal on-line biosensor for fed-batch culture. A stoichiometric growth reaction was formulated and data for material and heat fluxes incorporated into it. This showed that cell demand for glucose and glutamine was in the stoichiometric ratio of ∼3:1 rather than the ∼5:1 in the medium. It was demonstrated that the set of stoichiometric coefficients in the reaction were related through the extent of reaction (advancement) to overall metabolic activity (flux). The fact that this approach can be used for medium optimisation is the basis for an amino-acid-enriched medium which improved cell growth while decreasing catabolic fluxes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Short-term BOD (BODst) as a parameter for on-line monitoring of biological treatment process; Part II: instrumentation of integrated flow injection analysis (FIA) system for BODst estimation

An instrument with integrated flow injection analysis (FIA) system has been developed for on-line monitoring a process for conversion of biomass under field condition. The instrument consists of a newly designed biosensor for easy renewal of the bio-receptor without disassembling the sensor, a FIA controller for controlling the analysis operations, and a computer-based data acquisition system for data recording and processing. The instrument performed a sequence operations automatically including preparation of sample in the desired concentration, sample loading, sample injection, signal recording, data processing, and self-cleaning of the system. This makes the instrument being an interesting and promising device for on-line process monitoring.     

Correlating on-line monitoring parameters, pH, DO and ORP with nutrient removal in an intermittent cyclic process bioreactor system

The paper presents the study correlating the profile of on-line monitoring parameters and nutrient removal in an intermittent cyclic process bioreactor (ICPBR) system, thereby utilizing the parameters as operational tool. A laboratory scale ICPBR was employed to treat low C/N ratio domestic wastewater from a township. The study was conducted for correlating biological nutrient removal and on-line monitored parameters pH, dissolved oxygen (DO) and oxidation-reduction potential (ORP). The results revealed that pH, DO and ORP related with the dynamic behavior of nutrient concentration (NH4-N, NO3-N, and PO4-P) during treatment in an ICPBR system. The variation in pH and ORP of the reactor liquor correlate to conversion of ammonia (NH4-N) and nitrate (NO3-N) concentrations, respectively. As the bioconversion of ammonia nitrogen and phosphorus are related to the varying profile of the on-line monitored parameters, the profiles could possibly be used as onsite process control parameters.     

On-line monitoring of the methanogenic fermentation by measurement of culture fluorescence

Summary Real-time on-line fluorescence measurements of the coenzymes NAD(P)H and F₄₂₀ were evaluated as indicators of stability in a glucose-fed anaerobic methanogenic digester. A probe designed forin situ fluorimetric measurement of NAD(P)H provided an assessment of activity of the total microbial community, while the response of a fluorescence probe designed to measure coenzyme F₄₂₀ correlated well with methanogenic activity. The two fluorescence-monitoring probes responded directly to fermentation imbalance during periods of substrate overloading and corresponded to traditional offline measurements, suggesting that the probes may be suitable candidates for inclusion in an on-line process control system for anaerobic digestion.Florida Agricultural Experimental Station, Journal Series no. R-00326     

Some aspects of hybridoma cell cultivation

Summary Two hybridoma cell lines were cultivated in an indirectly aerated 10-1 reactor in batch, fed-batch and continuous (perfusion) operations and in spinner flasks. The medium in the reactor was sampled either by an aseptic cross-flow filtration module integrated into a loop or by an in-situ tubular filter. The glucose concentration was monitored by an on-line flow injection analyzer and the ammonia concentration by an ion-selective electrode. Since the membrane transmission of the high-molecular components decreased during cultivation, the product, a monoclonal antibody, was enriched in the reactor. During cultivation, the concentrations of cells, viable cells, glucose, lactase, acetate, citrate, ammonia, urea, amino acids, proteins, and monoclonal antibodies were determined off-line. The specific growth rate, specific production, and consumption rates of the medium components were influenced considerably by the medium composition, especially by the type and amount of serum used.Offprint requests to: K. Schügerl     

On-line monitoring of monoclonal antibody formation in high density perfusion culture using FIA

An automated flow injection system for on-line analysis of proteins in real fermentation fluids was developed by combining the principles of stopped-flow, merging zones flow injection analysis (FIA) with antigen-antibody reactions. IgG in the sample reacted with its corresponding antibody (a-IgG) in the reagent solution. Formation of insoluble immunocomplexes resulted in an increase of the turbidity which was determined photometrically. This system was used to monitor monoclonal antibody production in high cell density perfusion culture of hybridoma cells. Perfusion was performed with a newly developed static filtration unit equipped with hydrophilic microporous tubular membranes. Different sampling devices were tested to obtain a cell-free sample stream for on-line product anlysis of high molecular weight (e.g., monoclonal antibodies) and low molecular weight (e.g., glucose, lactate) medium components. In fermentation fluids a good correlation (coefficient: 0.996) between the FIA method and an ELISA test was demonstrated. In a high density perfusion cultivation process mAb formation was succesfully monitored on-line over a period of 400 h using a reliable sampling system. Glucose and lactate were measured over the same period of time using a commercially available automatic analyser based on immobilized enzyme technology.Abbreviations TIA Turbidimetric immunoassay - mAb Monoclonal Antibody     

Closed-loop control of fed-batch cultures of recombinant Escherichia coli using on-line HPLC

This article describes a fully automated system for the on-line monitoring and closed-loop control of a fed-batch fermentation of recombinant Escherichia coli, and presents two case studies of its used in limiting production of unwanted byproducts such as acetic in fed-batch fermentations. The system had two components. The first components, on-line monitoring, comprised an aseptic sampling device, a microcentrifuge, and HPLC System. These instruments removed a Sample from a fermentor, spun it at high speed to separate solid and liquid components, and then automatically injected the supernatant onto an HPLC column for analysis. The second component consisted of control algorithms programmed using the LabView visual programming environment in a control computer that was linked via a remote components were linked so that results from the on-line HPLC were captured and used by the control algorithm was designed to demonstrate coarse feedback control to confirm the operability of the controller. The second case study showed how the system could be used in a more sophisticated feedings strategy providing fine control and limiting acetate concentration to a low level throughout the fermentation. (c) 1994 John Wiley & Sons, Inc.     

Fluorimetric quantification of intracellular lactate dehydrogenase during fermentation using flow injection analysis

A flow injection system for the on-line detection of the intracellular enzyme lactate dehydrogenase (LDH) during fermentation has been developed. The system is comprised of an on-line cell disintegration part, an immobilised dye based expanded bed column for the affinity capture of LDH and a fluorimetric detection unit. The system with a linearity of 0.1–5.4 U LDH ml^–1 was applied for the detection of intracellular accumulation of LDH during Lactococcus lactis subsp.lactis cultivation.     

A reagentless amperometric electrode based on carbon paste, chemically modified with D-lactate dehydrogenase, NAD(+), and mediator containing polymer for D-lactic acid analysis. II. On-line monitoring of fermentation process

The production of D-lactic acid by Lactobacillus delbrueckii (ATCC 9649) during fermentation was monitored on-line with a reagentless D-lactate dehydrogenase modified carbon paste electrode in a flow injection system integrated with a filtration sampling device. The time delay between sampling and detection was approximately 6 min. The use of an electropolymerized ortho-phenylenediamine membrane on the elctrode resulted in a very selective sensor response with acceptable stability and sensitivity. The D-lactate concentrations determined on-line agreed well with those determined by a standard method, suggesting that this sensor system is suitable for on-line monitoring of fermentation processes. (c) 1995 John Wiley & Sons, Inc.     

Gas chromatography and gateway sensors for on-line state estimation of complex fermentations (butanol-acetone fermentation)

A fermentation system has been designed to demonstrate the use of gas chromatography (GC) for on-line monitoring of the butanol-acetone and other complex saccharolytic fermentations. Tangential flow ultrafiltration was used to sterilely and continuously obtain a cell-free filtrate from the fermentation broth for on-line GC analysis of butanol, butyrate, acetate, acetone, ethanol, and acetoin. The liquid injection system consists of a phosphoric acid contactor, a slider-type injection valve, and a heater to address the difficulties (ghosting) encountered in the analysis of carboxylic acids. The fermentation headspace gas was also analyzed by on-line GC for nitrogen and carbon dioxide, while hydrogen was measured by difference. Raw chromatographic data were analyzed by a chromatography data system. Both raw and processed data were transmitted to a VAX 11/750 computer for further processing (using the fermentation equation) and archiving. The fermentation equation, which has recently been derived and tested on completed fermentation data, was also found to be valid during transient fermentations and thus useful as a gateway sensor for calculating various fermentation parameters on-line. Such parameters include glucose concentration and gas composition, as well as a number of unobservable parameters (such as Y(ATP), excess ATP, and NAD reduced by FdH(2)), which characterize the state of the fermentation.     

On-line determination of intracellular beta-galactosidase activity in recombinant Escherichia coli using flow injection analysis (FIA).

A flow injection analysis (FIA) system was developed for the determination of cytoplasmic beta-galactosidase activity in recombinant Escherichia coli. The FIA system and its application for on-line monitoring of beta-galactosidase production during cultivation of recombinant E. coli in a 60-l airlift tower loop reactor is described. The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for on-line monitoring of intracellular protein formation.     

Biosensors for the Determination of Ortho-acetyl-L-carnitine. Their Utilization as Detectors in a Sequential Injection Analysis System

Abstract

In order to determine ortho-acetyl-L-carnitine, two biosensors were proposed. The biosensors were designed using physical immobilization of L-amino acid oxidase (L-AAOD) and horseradish peroxidase (HRP). Electrode characteristics were obtained and compared for the two carbon paste (graphite powder and paraffin oil) biosensors. The linear concentration ranges for the proposed biosensors were in the ranges of fmol/L to nmol/L, magnitude order with low limits of detection. Due to their reliability, the biosensors were used as detectors in a sequential injection analysis system, and gave reliable results for on-line assay of ortho-acetyl-L-carnitine in synthesis process control with a frequency of 75 samples per hour.     

Enzyme-based flow injection analysis system for glutamine and glutamate in mammalian cell culture media

We present the setup of a flow injection analysis system designed for on-line monitoring of glutamate and glutamine. These amino acids represent a major energy source in mammalian cell culture. A cycling assay consisting of glutamate dehydrogenase and aspartate aminotransferase produces NADH proportional to the glutamate concentration in the sample. NADH is then measured spectrophotometrically. Glutamine is determined by conversion to glutamate which is fed into the cycling assay. The conversion of glutamine to glutamate is catalyzed by asparaginase. Asparaginase was used in place of glutaminase due to its relatively high reactivity with glutamine and a pH optimum similar to that of glutamate dehydrogenase. The enzymes were immobilized covalently to activated controlled pore glass beads and integrated into the flow injection analysis system. The application of the immobilized enzymes and the technical setup are presented in this paper.     

A Contact-Imaging Based Microfluidic Cytometer with Machine-Learning for Single-Frame Super-Resolution Processing

Lensless microfluidic imaging with super-resolution processing has become a promising solution to miniaturize the conventional flow cytometer for point-of-care applications. The previous multi-frame super-resolution processing system can improve resolution but has limited cell flow rate and hence low throughput when capturing multiple subpixel-shifted cell images. This paper introduces a single-frame super-resolution processing with on-line machine-learning for contact images of cells. A corresponding contact-imaging based microfluidic cytometer prototype is demonstrated for cell recognition and counting. Compared with commercial flow cytometer, less than 8% error is observed for absolute number of microbeads; and 0.10 coefficient of variation is observed for cell-ratio of mixed RBC and HepG2 cells in solution.     

基于酶电极系统的葡萄糖浓度在线控制

补料分批技术在发酵工业中被广泛应用,其物料流加方式有3类,其中恒流速和指数补料属无反馈控制操作,靠经验或预设的数学模型决定补料速度,但由于发酵过程的复杂性,实际过程往往偏离预设的模型;恒底物浓度流加属反馈控制,通过对特定参数的检测,根据参数的变化情况反馈控制物料的流加,可控制菌生长在最佳条件下,从而获得高浓度的目的产物。反馈控制分直接控制和间接控制。间接     

Online naphazoline quality control by micellar‐enhanced spectrofluorimetry

The aim of this study was to develop a method for online spectrofluorimetric quality control of naphazoline (NPZ) in pharmaceuticals and raw drugs. A combination of a flow‐injection analysis (FIA) system with micellar‐enhanced fluorescence detection is presented as a powerful alternative for the rapid and sensitive analysis of naphazoline. Since NPZ shows low native fluorescence, the use of an anionic surfactant, such as sodium dodecyl sulphate (SDS), provides a considerable enhancement of fluorescence intensity and the nature of the technique allows a possible and easy adaptation to a FIA system. Using λ_exc = 280 nm and λ_em = 326 nm, a good linear relationship (LOL) was obtained in the range 0.003–10 µg mL^?1 with a detection limit (LOD) of 3 × 10^?4 µg mL^?1 (s/n = 3). Parameters related to the nature of the analytical signal and to the FIA manifold were optimized. Satisfactory recoveries were obtained in the analysis of commercial pharmaceutical formulations. The proposed method is simple, accurate and allows for high‐speed sampling and considerably shorter analysis times. In addition, it requires inexpensive equipment and reagents and has easy operational conditions and no side effects, thus avoiding environmental pollution through toxic waste. Copyright © 2011 John Wiley & Sons, Ltd.     

Use of a micro-expanded bed containing immobilised lysozyme for cell disruption in flow injection analysis

A method for cell disruption in Flow Injection Analysis (FIA) systems has been developed. The principle involves on-line cell disruption by means of immobilised lysozyme followed by an ultrasonic treatment. In order to avoid flow problems in the analytical system, the lysozyme was immobilised to Streamline^reg that was used in an expanded bed in the flow system. Samples of suspensions of Micrococcus lysodeikticus were treated and the success of the treatment was evaluated in terms of released protein and as a decrease in the optical density at 450 nm. The new technology offers a powerful tool in flow injection analyses for quantification of intracellular compounds. The concept of integration, i.e. combining cell disruption with handling of cell debris and assay procedure in one continuous flow process facilitates its use and increases the probability of reaching reproducible and reliable results.