Development of an automated flow-injection system for the determination of trace level ammonia

An in-housed designed computerised flow injection system for low level ammonia analysis is examined. The system features an on-line microdistillation preconcentration unit, which was used as an on-line sample pretreatment step in an ammonia gas-sensing probe flow injection system. A simple, low cost computerised control and data acquisition system was designed using a commercial pH meter with RS-232 interface and in-house designed control system. The system offered a practical and effective means of extending the detection limit of commercial available ammonia gas sensing probes to 5 μg/1 NH₃N.     

Exploring the recognized bio-mimicry materials for gas sensing

This study was undertaken to synthesize peptides that are partially similar to the binding sites of human olfactory receptor protein. First, a putative 3-D model structure of human olfactory receptor protein (P30953) was modeled using a molecular simulation method. The computer docking simulation was then performed to determine the most plausible binding sites between the model structure and target gases, trimethylamine, ammonia, acetic acid, and o-xylene. According to the simulation result, a series of polypeptide sequences, horp61 for TMA, horp103 for o-xylene, horp109 for ammonia, and horp193 for acetic acid as recognized molecules were designed for gas sensing purposes. Preparing these peptides as corresponding gas sensing probes, the results showed a high relative sensitivity response of 6.7 for TMA (probe horp61), 5.1 for o-xylene (probe horp103), 11 for ammonia (probe horp109), and 28 for acetic acid (probe horp193), respectively. These results indicate that peptide mimicking of binding domain on olfactory receptor opens a new window and offers a novel strategy for the further development of recognized materials for gas sensing.     

Surface Plasmon Resonance Based Fiber Optic Ammonia Sensor Utilizing Bromocresol Purple

In this paper, a surface plasmon resonance (SPR) based fiber optic ammonia gas sensor has been designed and fabricated using bromocresol purple (BCP) as sensing element. The sensor works under wavelength modulation scheme. The detection of ammonia gas has been carried out at room temperature. Three different kinds of film coating configurations, namely silver + BCP, gold + BCP, and silver + silicon + BCP on the unclad portion of the fiber have been used for studying the role of each layer. Further, to optimize the performance of the sensor, the films of varying thicknesses were coated using thermal evaporation technique. Experiments have been performed for the ammonia concentrations ranging from 0 to 150 ppm around the probe. To record the SPR spectrum, light from a polychromatic source is launched in the fiber and the spectrum is recorded at the other end of the fiber. The spectrum has a peak at lower wavelength while a dip at the higher wavelength. The dip corresponds to SPR while the peak appears to be due to fluorescence properties of the dye. It has been observed that as the ammonia gas comes in contact of the BCP layer, it changes the refractive index of the BCP dye which, in turn, changes the resonance wavelength. Further, the change in refractive index increases as the concentration of ammonia gas increases up to certain concentration of ammonia after that it saturates. Silicon layer has been shown as a protection layer for silver and gold from oxidation and acts as a tuner of wavelength. The proposed ammonia sensor has small response as well as recovery time.     

A protein recycling system for nuclear magnetic resonance-based screening of drug candidates

A sample-treating system for nuclear magnetic resonance (NMR)-based interaction screening between drug candidates (small molecules) and a protein of interest was developed by applying high-performance liquid chromatography (HPLC) technology. The system prepares a test solution by mixing a (15)N-labeled protein solution and a solution of each candidate compound, loads it to a flow cell-type NMR probe, and recycles the protein after the data acquisition. The system was designed to behave differently according to the information obtained in NMR measurements. In a test operation with a 100-compound library, the system could single out known interacting substances properly. Recovery values of the protein and one representative compound were 75 and 71%, respectively, and the recovered protein was found intact as intended.     

Flow injection analysis of serum urea using urease covalently immobilized on 2-fluoro-1-methylpyridinium salt-activated fractogel and fluorescence detection

Serum samples were analyzed for their urea content using fluorescence flow injection analysis incorporating an immobilized urease bioreactor and a gas permeable separator. The urease was immobilized under mild and facile conditions to a hydrophilic 2-fluoro-1-methylpyridinium-activated support. The ammonia released as a result of urease-catalyzed urea hydrolysis diffused through a gas permeable membrane into a constant stream of o-phthaldehyde solution to form a highly fluorescent product with lambda ex at 340 nm and lambda em at 455 nm. Up to 25 serum samples can be analyzed per hour. The within-day coefficient of variation (CV) was 1.12% and the day-to-day CV was 1.25% for serum containing 10.50 mg urea nitrogen dl-1. The bioreactor shows excellent storage (at 4 degrees C) and operational stabilities (at 37 degrees C).     

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.     

Sensory acquisition in active sensing systems

A defining feature of active sensing is the use of self-generated energy to probe the environment. Familiar biological examples include echolocation in bats and dolphins and active electrolocation in weakly electric fish. Organisms that utilize active sensing systems can potentially exert control over the characteristics of the probe energy, such as its intensity, direction, timing, and spectral characteristics. This is in contrast to passive sensing systems, which rely on extrinsic energy sources that are not directly controllable by the organism. The ability to control the probe energy adds a new dimension to the task of acquiring relevant information about the environment. Physical and ecological constraints confronted by active sensing systems include issues of signal propagation, attenuation, speed, energetics, and conspicuousness. These constraints influence the type of energy that organisms use to probe the environment, the amount of energy devoted to the process, and the way in which the nervous system integrates sensory and motor functions for optimizing sensory acquisition performance.     

Nitrogen removal reactor using packed gel envelopes containing Nitrosomonas europaea and Paracoccus denitrificans

Packed gel envelopes were constructed as simple, compact reactors for removing nitrogen from wastewater. Each packed gel envelope consisted of two plate gels with a spacer in between. Nitrosomonas europaea and Paracoccus denitrificans were co-immobilized in the plate gels, and ethanol, serving as an electron donor for denitrification, was injected into the internal spaces of the envelopes. The external surfaces of the envelopes were in contact with ammonia-containing wastewater; the N. europaea present in the gels oxidized the ammonia to nitrite aerobically. On the other hand, the internal surfaces of the envelopes were in contact with the ethanol solution, which P. denitrificans used to reduce the nitrite to nitrogen gas anaerobically. In this way, the reactor using the packed gel envelopes removed ammonia from wastewater in a single step. When artificial wastewater containing 200 mg-N/L was treated using the reactor using eight envelopes, the ammonia was removed by the reactor without accumulating nitrite or ethanol. This simple system exhibited high rates of nitrification (ammonia to nitrite; 1.9 kg-N/day for 1m(3) of reactor volume) and nitrogen removal (ammonia to nitrogen gas; 1.6 kg-N/day). It is presumed that these high rates were achieved as a consequence of cooperation between the N. europaea and P. denitrificans present in the gels and the efficient uptake and exhaust of gases leading to the smooth conversion of ammonia to nitrogen gas.     

A helium gas probe for use in cryosurgery

The design and testing of a prototype cryosurgical probe utilizing helium gas precooled with liquid nitrogen are described. An 8-mm-diameter probe produced an ice ball with a diameter of 28 mm after 10 min freezing using a helium gas flow rate of 42 liter/min. This indicated a surface heat transfer coefficient of 0.34 W/cm² °K and temperature of ?138 °C at the probe tip. Improved performance figures can be achieved using higher gas pressures and flow rates. A helium gas flow system schematic for use with this new type of cryoprobe is also presented. It is claimed that this system will overcome the problems of developing both multiple-tipped probes and small-diameter needle probes for use in cryoanalgesia.     

Squeezed flow preconcentration for probe tip biosensors

The preconcentration of analytes improves sensing using probe tips. In this work, we report a method based on creating a squeeze flow between a cylinder and circular coverslip to preconcentrate material at the liquid–gas interface while allowing a probe tip to be readily inserted there. In verification tests using enhanced green fluorescent protein, this capacity is proven. We estimated a 9.7 times increase in probability for fluorophores to be picked up at the tip using inference from fluorescence intensity distributions found. The method is expeditious, simple, and inexpensive, and it does not require any electrical energy source to operate.     

Modeling of local dynamic behavior of phenol degradation in an internal loop airlift bioreactor by yeast Candida tropicalis

A coupled computational fluid dynamic (CFD) model, combining hydrodynamics with biochemical reactions, was developed to simulate the local transient flow patterns and the dynamic behaviors of cell growth and phenol biodegradation by yeast Candida tropicalis in an internal loop airlift reactor (ILALR). To validate this proposed model effectively, the simulated local hydrodynamic characteristics of the gas-mineral salt medium solution (gas-liquid) two-phase system, at a phenol concentration of 1,200 mg L(-1) and no presence of cells, was experimentally investigated in the ILALR using laser Doppler anemometer (LDA) measurements and conductivity probe. Furthermore, the validation of the simulated phenol biodegradation behavior by C. tropicalis at different initial concentrations of phenol and cell was also carried out in the ILALR. The time-averaged and transient results of the model simulations illustrated a satisfactory agreement with the experimental data. Finally, the local instantaneous flow and phenol biodegradation features, including gas holdup, gas velocity, liquid velocity, cell concentration, and phenol concentration inside the ILALR were successfully predicted by the proposed model.     

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.     

Green synthesis of biopolymer-silver nanoparticle nanocomposite: An optical sensor for ammonia detection

Biopolymer used for the production of nanoparticles (NPs) has attracted increasing attention. In the presence article we use aqueous solution of polysaccharide Cyamopsis tetragonaloba commonly known as guar gum (GG), from plants. GG acts as reductive preparation of silver nanoparticles which are found to be <10nm in size. The uniformity of the NPs size was measured by the SEM and TEM, while a face centered cubic structure of crystalline silver nanoparticles was characterized using powder X-ray diffraction technique. Aqueous ammonia sensing study of polymer/silver nanoparticles nanocomposite (GG/AgNPs NC) was performed by optical method based on surface plasmon resonance (SPR). The performances of optical sensor were investigated which provide the excellent result. The response time of 2-3s and the detection limit of ammonia solution, 1ppm were found at room temperature. Thus, in future this room temperature optical ammonia sensor can be used for clinical and medical diagnosis for detecting low ammonia level in biological fluids, such as plasma, sweat, saliva, cerebrospinal liquid or biological samples in general for various biomedical applications in human.     

Nitrification and anammox with urea as the energy source

Urea is present in many ecosystems and can be used as an energy source by chemolithotrophic aerobic ammonia oxidizing bacteria (AOB). Thus the utilization of urea in comparison to ammonia, by AOB as well as anaerobic ammonia oxidizing (Anammox) bacteria was investigated, using enrichments cultures, inoculated with activated sludge, and molecular ecological methods. In batch enrichment cultures grown with ammonia a population established in 2 weeks, which was dominated by halophilic and halotolerant AOB as determined by fluorescence in situ hybridization (FISH) experiments, with the 16S rRNA targeting oligonucleotide probe NEU. In other batch enrichment cultures using urea, the AOB population was assessed by PCR amplification, cloning and phylogenetic analysis of amoA and ribosomal 16S rRNA genes. While only one of the 48 16S rRNA gene clones could be identified as AOB (Nitrosomonas oligotropha), the amoA approach revealed two more AOB, Nitrosomonas europaea and Nitrosomonas nitrosa to be present in the enrichment. FISH analysis of the enrichment with probe NEU and newly designed probes for a specific detection of N. oligotropha and N. nitrosa related organisms, respectively, showed that N. oligotropha-like AOB formed about 50% of the total bacterial population. Also N. nitrosa (about 15% of the total population) and N. europaea (about 5% of the total population) were relatively abundant. Additionally, continuous enrichments were performed under oxygen limitation. When ammonia was the energy source, the community in this reactor consisted of Anammox bacteria and AOB hybridizing with probe NEU. As the substrate was changed to urea, AOB related to N. oligotropha became the dominant AOB in this oxygen limited consortium. This resulted in a direct conversion of urea to dinitrogen gas, without the addition of organic carbon.     

Micro analysis system for pH and protease activities with an integrated sample injection mechanism

A micro analysis system for the electrochemical determination of the activity of protease along with pH sensing was fabricated aiming for its use in telemetric micro analysis systems targeting the testing of the stomach and intestines. The system consisted of a pH-sensing site and two protease assay sites formed in polydimethylsiloxane (PDMS) micro flow channels. To introduce sample solutions, valves were formed with gold electrodes in the inlets, which functioned on the basis of electrowetting. An external sample solution could be introduced into the sensing sites by switching on the valves at appropriate times. In the pH-sensing site, a pH-indicator electrode changed its electrode potential immediately after a sample solution reached an internal liquid-junction reference electrode. The slope of the calibration plot was -74.5 mVpH(-1). Bovine serum albumin (BSA) was used as the substrate for the enzyme and was spotted on the wall of the flow channel that faced the pH-indicator electrode of the protease assay sites. The release of protons accompanying the hydrolysis of BSA by the enzyme was detected using the pH-indicator electrode. When trypsin was contained in the sample solution as a test enzyme, a distinct decrease in pH, which was dependent on the trypsin activity, was observed, indicating that enzymatic hydrolysis was proceeding. The initial rate of potential change varied in proportion to the activity in a range between 1.0 and 51.7 Uml(-1). The integration of the microfluidic and sensing functions provides significant advantages for the use of this system as an isolated telemetric micro system that might operate with small batteries.     

Use of microbial electrodes for observation of microbiological nitrogen elimination process

Microbial electrodes for the determination of ammonia and the estimation of biochemical oxygen demand (BOD) were applied to the nitrogen elimination process. The dimensions of the nitrification and the denitrification vessels were 170 and 70 L, respectively. The wastewater used for the experiment was obtained from a fermentation factory and adjusted to 470-530 mg/L of total Kjeldahl nitrogen and 1700-3000 mg/L of BOD. The ammonia electrode was assembled with a membrane containing nitrifying bacteria and an oxygen probe. The BOD electrode was similarly constructed, except it used a membrane containing the yeast, Trichosporon cutaneum. A flow system was employed for the automatic measurement of samples every 30 min. The nitrification and denitrification rates of the activated sludge were measured to investigate the optimum conditions and evaluate the capacity of the plant. The various data obtained by the microbial electrodes allowed us to inspect the situation of the plant and estimate control paramerers such as nitrogen and BOD loadings. The average removals of ammonia nitrogen and total Kjeldahl nitrogen were 96% and 89%, respectively, during the experiment for period of 2 weeks.     

Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate

Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the submicromolar range was developed.     

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.     

Determination of ammonium and L-Glutamine in hybridoma cell cultures by sequential flow injection analysis

A flow injection anlytical system based on a gas diffusion membrane module for ammonia and an ammonium flow-through potentiometric detector has been set up for measurement of L-glutamine and ammonium ions in hybridoma cell cultures. The main feature of the system is that the same basic analytical concept and equipment is used in both measurements, the only difference being for the determination of L-glutamine, in which the sample flows through an immobilized glutaminase cartridge. The conditions to enable the performance of both analysis consecutively, avoiding potential interferences by unwanted deamination of other compounds in the samples, have been determined. Finally, the proposed system has been compared with reference analytical methods for batch hybridoma cell culture experiments.