Neuro-genetic optimization of temperature control for a continuous flow polymerase chain reaction microdevice

In this study, a hybridized neuro-genetic optimization methodology realized by embedding finite element analysis (FEA) trained artificial neural networks (ANN) into genetic algorithms (GA), is used to optimize temperature control in a ceramic based continuous flow polymerase chain reaction (CPCR) device. The CPCR device requires three thermally isolated reaction zones of 94 degrees C, 65 degrees C, and 72 degrees C for the denaturing, annealing, and extension processes, respectively, to complete a cycle of polymerase chain reaction. The most important aspect of temperature control in the CPCR is to maintain temperature distribution at each reaction zone with a precision of +/-1 degree C or better, irrespective of changing ambient conditions. Results obtained from the FEA simulation shows good comparison with published experimental work for the temperature control in each reaction zone of the microfluidic channels. The simulation data are then used to train the ANN to predict the temperature distribution of the microfluidic channel for various heater input power and fluid flow rate. Once trained, the ANN analysis is able to predict the temperature distribution in the microchannel in less than 20 min, whereas the FEA simulation takes approximately 7 h to do so. The final optimization of temperature control in the CPCR device is achieved by embedding the trained ANN results as a fitness function into GA. Finally, the GA optimized results are used to build a new FEA model for numerical simulation analysis. The simulation results for the neuro-genetic optimized CPCR model and the initial CPCR model are then compared. The neuro-genetic optimized model shows a significant improvement from the initial model, establishing the optimization method's superiority.     

Xenobiotic substrate reduces yield of activated sludge in a continuous flow system

The biomass yield of a continuous flow activated sludge system varied when the system treated influent containing different compositions of biogenic and xenobiotic substrates. Both the biogenic substrate and a test xenobiotic 2,4-dichlorophenoxyacetic acid (2,4-D) were degraded at steady-state activated sludge operations. The true yields, determined from steady-state activated sludge treatment performances, were at the maximum and the minimum when the activated sludge treated the influent of sole biogenic substrate and sole 2,4-D, respectively. The minimum yield was 56% of the maximum. Yield reduction between the maximum and the minimum was proportional to the concentration of 2,4-D in the influent. This trend of yield reduction suited a model that describes the metabolic uncoupling effect of 2,4-D on the sludge's degradation of the substrates. The model function variable was defined as the ratio of 2,4-D to biogenic COD concentrations in the influent.     

Kinetic study of immobilized chloroplast membranes operating in a continuous flow system

We have focused our interest on the study of the operational stability of immobilized chloroplast membranes. Due to the physical insoluble form of immobilized material this study can be done in a continuous flow system. The activity of immobilized chloroplast membranes is monitored continuously during the inactivation treatment and modelled under operating conditions by taking into account concomitant inactivation with time. Theoretical and experimental results are compared. The effect of light conditions on the operational stability is also reported.     

An electrochemical immunosensor for milk progesterone using a continuous flow system

An electrochemical biosensor for cow's milk progesterone has been developed and used in a competitive immunoassay under thin-layer, continuous-flow conditions. Single-use biosensors were fabricated by depositing anti-progesterone monoclonal antibody (mAb) onto screen-printed carbon electrodes (SPCEs). Three operational steps could be identified: (1) Competitive binding of sample/conjugate (alkaline-phosphatase-labelled progesterone, AP-prog) mixture, (2) establishment of a steady-state amperometric baseline current and (3), measurement of an amperometric signal in the presence of enzyme substrate (1-naphthyl phosphate, 1-NP). In the thin-layer cell, the enzyme product, 1-naphthol, showed electrochemical behaviour consistent with bulk conditions and gave a linear amperometric response under continuous-flow conditions (E_app=+0.3 V vs. Ag/AgCl) over the range 0.1–1.0 μg/ml. After pre-incubating biosensors with progesterone standards, signal generation within the cell (substrate CONCENTRATION=5 mM) was recorded amperometrically as rate (nA/s) or maximum current (i_max, nA). Response values for milk standards were approximately 50% of those prepared in buffer. In both cases, calibration plots over the range 0–50 ng/ml progesterone were obtained. By conducting sample binding under flowing conditions, only 7% of the previous response was obtained, even at a substrate concentration of 50 mM, resulting in low signal:noise ratio. Using a stop-flow arrangement (i.e. quiescent sample binding, followed by continuous flow), low-noise amperograms were obtained at [1-NP]=5 mM. Calibration plots were obtained over the range 0–25 ng/ml, with a coefficient of variation of 12.5% for five replicate real milk samples.     

Radionuclide accumulation by Anodonta piscinalis Nilsson (Lamellibranchiata) in a continuous flow system

Summary The present investigation has been concerned with the accumulation of radionuclides by the freshwater lamellibranch Anodonta piscinalis Nilsson. Specimens of the animal were exposed for 125 days to approximately constant concentrations of the radionuclides P³², Sr⁸⁹, Cs¹³⁷, Ce¹⁴⁴ and Zr⁹⁵/Nb⁹⁵; in addition Ru¹⁰³ was included among the radionuclides for the last 55 days of the experiment. The investigation was conducted in a model recipient with environmental factors similar to those existing in the animals' biotope in nature. The accumulation of the radionuclides was measured in the body and valves of the lamellibranch. The results indicate that an approximately constant level of radioactivity was reached after one month's exposure in these two parts of the animal. Subsequently the level of radioactivity showed a fluctuating pattern which was ascribed to seasonal and physiological changes. Accumulation of the radionuclides in the test animals varied according to the following sequence, arranged in order of decreasing affinity: P³² > Sr⁸⁹ > Ce¹⁴⁴ > Ru¹⁰³ > Cs¹³⁷ > Zr⁹⁵/Nb⁹⁵. Factors which may complicate the practical use of freshwater lamellibranchs in the monitoring of contaminated recipients are pointed out and discussed.

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Zusammenfassung Die vorliegende Untersuchung betrifft die Anh?ufung von Radioisotopen bei der Lamellibranchiate Anodonta piscinalis Nilsson. Exemplare dieses Tieres wurden beobachtet w?hrend 125 Tagen bei ann?hernd konstanter Konzentration der Isotopen P³², Sr⁸⁹, Cs¹³⁷, Ce¹⁴⁴ und Zr⁹⁵/Nb⁹⁵ w?hrend 55 Tagen des Experimentes wurde Ru¹⁰³ miteinbezogen. Das Experiment wurde in einem Flussmodell durchgeführt bei gleichen Umgebungsfaktoren wie sie das Tier in seinem natürlichen Biotop findet. Die Anh?ufung der radioaktiven Isotope wurde im K?rper und in der Schale der Lamellibranchiate gemessen. Das Resultat erwies, dass innerhalb eines Monats das Tier eine gewisse Radioaktivit?t erreichte in den zwei geprüften Teilen. Das Radioaktivit?tsniveau wies eine wellenartige Schwankung auf, welche dem Jahreszeitlichen und physiologischen Wechsel zuzuschreiben ist. Die Radioisotope wurden im Tier in folgender Reihenfolge angeh?uft: P³² > Sr⁸⁹ > Ce¹⁴⁴ > Ru¹⁰³ > Cs¹³⁷ > Zr⁹⁵/Nb⁹⁵. Faktoren komplizierter Natur sind für den praktischen Gebrauch der Süsswasserlamellibranchiata in der Radioaktivit?ts überwachung verunreinigter Gew?sser begrenzend, und wurden hier diskutiert.

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This work was performed under International Atomic Energy Agency Research Contract No. 37.     

Inactivation of enzymes in fresh sake using a continuous flow system for high-pressure carbonation

The Inactivation kinetics of alpha-glucosidase, glucoamylase, alpha-amylase, and acid carboxypeptidase in fresh sake using a continuous flow system for high-pressure carbonation were investigated. In addition, the effects of ethanol and sugar concentrations on inactivation of the enzymes in high-pressure carbonated sake were investigated. Among the enzymes investigated, alpha-glucosidase was the most stable and alpha-amylase was the most labile on inactivation under carbonation. The decimal reduction times (D values) of alpha-glucosidase, glucoamylase, alpha-amylase (extrapolated from the Z value), and acid carboxypeptidase were 29, 6, 2, and 5 min respectively at 45 degrees C. These values are lower than those subjected to heat treatment. On the carbonation treatment as well as the heat treatment, ethanol accelerated the inactivation of all four enzymes, but glucose depressed the inactivation of these enzymes, except for acid carboxypeptidase. These results suggest that this continuous flow system enabled effective inactivation of enzymes in fresh sake.     

Meningococcal biofilm formation: structure, development and phenotypes in a standardized continuous flow system

We show that in a standardized in vitro flow system unencapsulated variants of genetically diverse lineages of Neisseria meningitidis formed biofilms, that could be maintained for more than 96 h. Biofilm cells were resistant to penicillin, but not to rifampin or ciprofloxacin. For some strains, microcolony formation within biofilms was observed. Microcolony formation in strain MC58 depended on a functional copy of the pilE gene encoding the pilus subunit pilin, and was associated with twitching of cells. Nevertheless, unpiliated pilE mutants formed biofilms showing that attachment and accumulation of cells did not depend on pilus expression. Mutation and complementation analysis revealed that the type IV pilus-associated protein PilX, which was recently shown to mediate interbacterial aggregation, indirectly supported microcolony formation by contributing to pilus expression. A large number of PilX alleles was identified among genetically diverse meningococcal strains. PilX alleles differed in their propensity to support autoaggregation of cells in suspension, but not in their ability to support microcolony formation within biofilms in the continuous flow system.     

Possible complication regarding phosphorus removal with a continuous flow biofilm system: diffusion limitation

Diffusion limitation of phosphate possibly constitutes a serious problem regarding the use of a biofilm reactor for enhanced biological phosphorus removal. A lab-scale reactor for simultaneous removal of phosphorus and nitrate was operated in a continuous alternating mode of operation. For a steady-state operation with excess amounts of carbon source (acetate) during the anaerobic phase, the same amount of phosphate was released during the anaerobic phase as was taken up during the anoxic phase. The measured phosphorus content of the biomass that detached during backwash after an anoxic phase was low, 2.4 +/- 0.4% (equal to 24 +/- 4 mg P/g TS). A simplified computer model indicated the reason to be phosphate diffusion limitation and the model revealed a delicate balance between the obtainable phosphorus contents of the biomass and operating parameters, such as backwash interval, biofilm thickness after backwash, and phase lengths. The aspect of diffusion is considered of crucial importance when evaluating the performance of a biofilter for phosphate removal.     

Production of an enzymatic active protein using a continuous flow cell-free translation system.

We have examined the characteristics of protein synthesis in an improved continuous flow cell-free translation system prepared from wheat germ extract with dihydrofolate reductase (dhfr) mRNA as the translated message. Continuous buffer flow and separation of product from the reaction mixture were accomplished by the use of a modified Amicon ultrafiltration chamber as reaction vessel. The system produced protein for more than 20 h, and the product had an activity of dhfr comparable to that of authentic enzyme from E. coli. Analysis of RNA recovered from the filtrate supports the notion that a functionally active protein-synthesizing machinery is superorganized in a dynamic complex.     

A continuous flow automatically recording respirometer

A respirometer suitable for continuous operation over extended periods of time is described. Oxygen is supplied to the culture by the electrolysis of water. The quantity of oxygen produced by electrolysis within any specified time interval is automatically printed on a tape. This record is combined with a continuous recording of the concentration of dissolved oxygen entering and leaving the reactor to produce a mass balance for oxygen in the system. Details on respirometer construction and operation are presented.     

An immobilized alpha-galactosidase continuous flow reactor

An α-galactosidase which will hydrolyze the oligosaccharides melibiose, raffinose, and stachyose has been immobilized on nylon microfibrils suitable for use in large flow-through reactors. This catalyst system is stable for many months, both under use and storage conditions. The immobilized enzyme behaves similarly to the enzyme in solution, characteristically exhibiting both product and substrate inhibition. The catalyst is prepared in situ and a large, 8-liter reactor has been made. The catalyst has been used to reduce the raffinose concentration in beet sugar molasses.     

Evaluation of protein disulfide conversion in vitro using a continuous flow dialysis system

Recombinant therapeutic proteins are heterogeneous due to chemical and physical modifications. Understanding the impact of these modifications on drug safety and efficacy is critical for optimal process development and for setting reasonable specification limits. In this study, we describe the development of an in vitro continuous flow dialysis system to evaluate potential in vivo behavior of thiol adducted species and incorrectly disulfide bonded species of therapeutic proteins. The system is capable of maintaining the low-level cysteine concentrations found in human blood. Liabilities of cysteamine adducted species, incorrectly disulfide bonded species, and the correctly disulfide bonded form of an Fc-fusion protein were studied using this system. Results showed that 90% of the cysteamine adduct converted into the correctly disulfide bonded form and incorrectly disulfide bonded species in approximately 4 h under physiological conditions. Approximately 50% of incorrectly disulfide bonded species converted into the correctly bonded form in 2 days. These results provide valuable information on potential in vivo stability of the cysteamine adduct, incorrectly disulfide bonded species, and the correctly bonded form of the Fc-fusion protein. These are important considerations when evaluating the criticality of product quality attributes.     

Studying the phosphorus release from the Loosdrecht Lakes sediments,using a continuous flow system

Phosphorus release from the Loosdrecht Lakes sediments was studied, using a continuous flow reactor. The summer release maxima were 4 mg P.m^–2.d^–1 in 1984 and 1.4 mg P.m^–2.d^–1 in 1985. Temperature and downward seepage controlled release rates to a great extent, the pH of the overlying water being only of minor importance. From these results it could be concluded that release processes might be driven by mineralization of particulate organic phosphorus in the sediment. Pore water studies in the sediments of the release reactor confirmed this hypothesis. From the profiles phosphorus dissolution rates were calculated.