The effect of fluidic conditions on the continuous-flow bioluminescent detection of ATP in a microfluidic device

The effect of fluidic conditions on the bioluminescent detection of ATP in a microfluidic device was surveyed using homemade detector system. The bioluminescent reaction of ATP was directly affected by the retention time and flow rates of the solutions in this diffusion-based mixing and reaction system due to the laminar flow in the microchannel. ATP and enzyme solutions were separately injected into the microfluidic device at different flow rates through 3 inlet ports. The ATP solution was injected through the middle port, while the enzyme solution was injected in the two remaining ports. When the ratio of ATP to enzyme solution was fixed, the optimum flow rates of enzyme, ATP, and enzyme solution was 3.5, 8.0, and 3.5 μL/min, respectively. The optimal total flow rate was 15 μL/min. The detection limit for the concentration of ATP at optimal conditions was less than 10⁻⁷ M.     

Highly selective amperometric glucose microdevice derived from diffusion layer gap electrode

Although most of enzyme catalytic reactions are specific, the amperometric detection of the enzymatic reaction products is largely nonselective. How to improve the detection selectivity of the enzyme-based electrochemical biosensors has to be considered in the sensor fabrication procedures. Herein, a highly selective amperometric glucose biosensor based on the concept of diffusion layer gap electrode pair which we previously proposed was designed. In this biosensor, a gold tube coated with a conductive layer of glucose oxidase/Nafion/graphite was used to create an interference-free region in its diffusion layer by electrochemically oxidizing the interfering electroactive species at proper potentials. A Pt probe electrode was located in this diffusion layer of the tube electrode to selectively detect hydrogen peroxide generated from the enzyme catalytic oxidation of glucose in the presence of oxygen in the solution. In practical performance of the microdevice, parameters influencing the interference-removing efficiency, including the tip-tube opening distance, the tube electrode potential and the electrolyzing time had been investigated systematically. Results showed that glucose detection free from interferents could be achieved at the electrolyzing time of 30s, the tip-tube opening distance of 3mm and the tube electrode potential of 0.4V. The electrochemical response showed linear dependence on the concentration of glucose in the range of 1 x 10(-5) to 4 x 10(-3) M (the correlation coefficient: 0.9936, without interferents; 0.9995, with interferents). In addition, the effectiveness of this device was confirmed by numerical simulation using a model system of a solution containing interferents. The simulated results showed good agreement with the experimental data.     

Analysis of the multicopper oxidase gene regulatory network of Aeromonas hydrophila

Multicopper oxidase (MCO) is an enzyme which involves in reducing the oxygen in a four electron reduction to water with concomitant one electron oxidation of reducing the substrate. We have generated the 3-D structure of MCO by homology modeling and validated on the basis of free energy while 90.4 % amino acid residues present in allowed regions of Ramachandran plot. The screening of potential hazardous aromatic compounds for MCO was performed using molecular docking. We obtained Sulfonaphthal, Thymolphthalein, Bromocresol green and Phloretin derivatives of phenol and aromatic hydrocarbon were efficient substrates for MCO. The phylogeny of MCO reveals that other bacteria restrain the homologous gene of MCO may play an important role in biodegradation of aromatic compounds. We have demonstrated the gene regulatory network of MCO with other cellular proteins which play a key role in gene regulation. These findings provide a new insight for oxidization of phenolic and aromatic compounds using biodegradation process for controlling environmental pollution.     

Optimized lipase-catalyzed synthesis of adipate ester in a solvent-free system

Immobilized Candida antarctica lipase-catalyzed esterification of adipic acid and oleyl alcohol was investigated in a solvent-free system (SFS). Optimum conditions for adipate ester synthesis in a stirred-tank reactor were determined by the response surface methodology (RSM) approach with respect to important reaction parameters including time, temperature, agitation speed, and amount of enzyme. A high conversion yield was achieved using low enzyme amounts of 2.5% w/w at 60°C, reaction time of 438 min, and agitation speed of 500 rpm. The good correlation between predicted value (96.0%) and actual value (95.5%) implies that the model derived from RSM allows better understanding of the effect of important reaction parameters on the lipase-catalyzed synthesis of adipate ester in an organic solvent-free system. Higher volumetric productivity compared to a solvent-based system was also offered by SFS. The results demonstrate that the solvent-free system is efficient for enzymatic synthesis of adipate ester.     

A Comparison of Five Experimental Techniques to Measure Charge Carrier Lifetime in Polymer/Fullerene Solar Cells

It is important to accurately measure the charge carrier lifetime, a crucial parameter that influences the collection efficiency in organic solar cells. Five transient and small perturbation experimental techniques that measure charge carrier lifetime are applied to a device composed of the polymer PDTSiTTz blended with the fullerene PCBM: time‐resolved charge extraction (TRCE), transient absorption spectroscopy (TAS), photoinduced charge extraction by linearly increasing voltage (photo‐CELIV), transient photovoltage, and electrochemical impedance spectroscopy. The motivation is to perform a comprehensive comparison of several different lifetime measurement techniques on the same device in order to assess their relative accuracy, applicability to operational devices, and utility in data analysis. The techniques all produce similar charge carrier lifetimes at high charge densities, despite previous suggestions that transient methods are less accurate than small perturbation ones. At lower charge densities an increase in the apparent reaction order is observed. This may be related to surface recombination at the contacts beginning to dominate, or an inhomogeneous charge distribution. A combination of TAS and TRCE appears suitable. TAS enables the investigation of recombination mechanisms at early times since it is not limited by RC (resistance‐capacitance product) or charge extraction losses. Conversely, TRCE is useful particularly at low densities when other mechanisms, such as surface recombination, may occur.     

Microfluidic conductimetric bioreactor

A microfluidic conductimetric bioreactor has been developed. Enzyme was immobilized in the microfluidic channel on poly-dimethylsiloxane (PDMS) surface via covalent binding method. The detection unit consisted of two gold electrodes and a laboratory-built conductimetric transducer to monitor the increase in the conductivity of the solution due to the change of the charges generated by the enzyme-substrate catalytic reaction. Urea–urease was used as a representative analyte-enzyme system. Under optimum conditions urea could be determined with a detection limit of 0.09 mM and linearity in the range of 0.1–10 mM (r = 0.9944). The immobilized urease on the microchannel chip provided good stability (>30 days of operation time) and good repeatability with an R.S.D. lower than 2.3%. Good agreement was obtained when urea concentrations of human serum samples determined by the microfluidic flow injection conductimetric bioreactor system were compared to those obtained using the Berthelot reaction (P < 0.05). After prolong use the immobilized enzyme could be removed from the PDMS microchannel chip enabling new active enzyme to be immobilized and the chip to be reused.     

Effect of agitation on growth and enzyme production of Trichoderma reesei in batch fermentation

Growth, enzyme-producing activity and respiratory properties of Trichoderma reesei QM 9414 were examined under various agitation intensities. Two substrates were compared: lactose and Avicel. Pellet formation occurred at all agitation intensities for both substrates. Oxygen dependence at the lower agitation rate varied with the substrate type. With lactose as the carbon source, linear growth was observed, despite a regulation of the dissolved oxygen concentration at 30% saturation. The enzyme production was strongly affected by the agitation. At the higher agitation rates the enzyme production dropped. With Avicel as the carbon source, the production of enzymes surged as soon as the growth was limited by the hydrolysis of Avicel.Growth on Avicel, in the conditions we used, was limited by Avicel hydrolysis. Cubic growth was observed when lactose was the carbon source. A new derivation for a model of the observed cubic growth is proposed and is used to correlate growth, CO₂ production and oxygen consumption in a consistent way, impossible with exponential growth models.     

Electrochemical assay of endo-depolymerase activity of cellulases

A method for determination of endo-1,4-beta-D-glucanase activity of cellulase samples based on the indirect measurement of decrease in viscosity of a carboxymethylcellulose solution in an electrochemical cell in the presence of an electron carrier was developed. A rotating disk electrode is used as the working electrode. When two reactions (enzymatic and electrochemical) proceeded in the cell simultaneously, the limiting diffusion current at a constant applied potential increases as the viscosity of the solution decreases. Conditions where the initial rate of change of diffusion current (dI/dt) is proportional to the enzyme concentration were found. A good correlation between the new method and a previously known viscometric method for determination of endoglucanase activity was observed.     

Taste and smell GPCRs in the lung: Evidence for a previously unrecognized widespread chemosensory system

Taste and smell receptor expression has been traditionally limited to the tongue and nose. We have identified bitter taste receptors (TAS2Rs) and olfactory receptors (ORs) on human airway smooth muscle (HASM) cells. TAS2Rs signal to PLCβ evoking an increase in [Ca^2 +]_i causing membrane hyperpolarization and marked HASM relaxation ascertained by single cell, ex vivo, and in vivo methods. The presence of TAS2Rs in the lung was unexpected, as was the bronchodilatory function which has been shown to be due to signaling within specific microdomains of the cell. Unlike β₂-adrenergic receptor-mediated bronchodilation, TAS2R function is not impaired in asthma and shows little tachyphylaxis. HASM ORs do not bronchodilate, but rather modulate cytoskeletal remodeling and hyperplasia, two cardinal features of asthma. We have shown that short chain fatty acids, byproducts of fermentation of polysaccharides by the gut microbiome, activate HASM ORs. This establishes a non-immune gut-lung mechanism that ties observations on gut microbial communities to asthma phenotypes. Subsequent studies by multiple investigators have revealed expression and specialized functions of TAS2Rs and ORs in multiple cell-types and organs throughout the body. Collectively, the data point towards a previously unrecognized chemosensory system which recognizes endogenous and exogenous agonists. These receptors and their ligands play roles in normal homeostatic functions, predisposition or adaptation to disease, and represent drug targets for novel therapeutics.     

Factors screening to statistical experimental design of racemic atenolol kinetic resolution via transesterification reaction in organic solvent using free Pseudomonas fluorescens lipase

As the (R )‐enantiomer of racemic atenolol has no β‐blocking activity and no lack of side effects, switching from the racemate to the (S )‐atenolol is more favorable. Transesterification of racemic atenolol using free enzymes investigated as a resource to resolve the racemate via this method is limited. Screenings of enzyme, medium, and acetyl donor were conducted first to give Pseudomonas fluorescens lipase, tetrahydrofuran, and vinyl acetate. A statistical design of the experiment was then developed using Central Composite Design on some operational factors, which resulted in the conversions of 11.70–61.91% and substrate enantiomeric excess (ee ) of 7.31–100%. The quadratic models are acceptable with R² of 95.13% (conversion) and 89.63% (ee ). The predicted values match the observed values reasonably well. Temperature, agitation speed, and substrate molar ratio factor have low effects on conversion and ee , but enzyme loading affects the responses highly. The interaction of temperature–agitation speed and temperature–substrate molar ratio show significant effects on conversion, while temperature–agitation speed, temperature–substrate molar ratio, and agitation speed–substrate molar ratio affect ee highly. Optimum conditions for the use of Pseudomonas fluorescens lipase, tetrahydrofuran, and vinyl acetate were found at 45°C, 175 rpm, 2000 U, and 1:3.6 substrate molar ratio.     

Studies on the production of bacterial rennet in a pilot plant fermentor

An attempt was made to find out the optimum aeration and agitation rates on the production of bacterial rennet from Bacillus sublilis K-26 using 5% wheat bran medium in a 13 liter fermentor. The enzyme activity and the growth rate were shown to increase with an increase in the rate of agitation. The fermentation experiments carried out at an agitation rate of 400 rpm showed an approximate threefold increase in enzyme activity with a considerable decrease in the fermentation time over those agitated at 200 and 300 rpm. The beneficial effect of a higher oxygen rate was observed for enzyme production occurring at a lower agitation rate. The inoculum activity and the varying amounts of antifoam agent which were added showed no apparent effect either on the total incubation time or on the final enzyme activity. It has been suggested that an agitation rate of 400 rpm with an aeration level of 3000 cc/min are the optimum values for the efficient production of bacterial rennet from B. subtilis K-26 using 5% wheat bran medium in a 13 liter fermentor.     

Detection of allergen specific immunoglobulins by microarrays coupled to microfluidics

Allergen microarrays are under development for a component‐resolved diagnosis of Type I (IgE‐mediated) allergies. Here we report an improved microarray coupled to microfluidics for the detection of allergen specific immunoglobulin E (IgE). The signal intensity for IgE detection in serum has been improved by using glass slides coated with a novel poly[DMA‐co‐NAS] brush copolymer which is able to immobilize allergens in their native conformation and by carrying out the incubation step in dynamic conditions. The assay, fully automated, was performed in a microcell, using a software‐controlled fluidic processor, to bring assay reagents on the surface of the array. Microfluidics turns the binding between serum immunoglobulins and immobilized allergens from a diffusion‐limited to a kinetic‐limited process by ensuring an efficient mixing of serum samples on the surface of the microarray. As a result of this, the binding of high affinity IgE antibodies is enhanced whereas that of low affinity IgG antibodies, which are present at higher concentration, is impaired paving the way to more accurate and sensitive results.     

Copper catalysed oxidation of amino acids and Alzheimer's disease

Summary Metal-catalyzed oxidation (MCO) can lead to damage of bio-molecules and is implicated in neurodegenerative diseases, such as Alzheimer's disease (AD). The amino acid residues, tyrosine, histidine and methionine, have been proposed to play important roles in metal mediated oxidative stress and subsequent reactions of amyloid β peptide (Aβ) a major contributor in the pathogenesis of AD. The MCO of Aβ residues, particularly histidine, methionine and tyrosine, and reviewed. MCO of Aβ histidine and tyrosine residues can facilitate oligomerization and may play a role in both amyloid formation and Aβ neurotoxicity. Further work is needed to determine the importance of Aβ oxidation in AD and the role of Aβ oxidation products and oxidative stress in disease progression. The mechanisms of Aβ MCO are complex and multiple reaction products can form. Further study is needed to determine the mechanisms by which Aβ MCO occursin vivo. In addition, new analytical methods are required to monitor the formation of Aβ MCO products formed during AD. The copper-H₂O₂ redox system provides a chemical model by which Aβ MCO can be studiedin vitro and can be used to produce oxidatively modified amino acid residues for use as standards in developing new analytical methods to monitor Aβ MCO.     

Development,validation and enzyme kinetic evaluation of multi walled carbon nano tubes mediated tyrosinase based electrochemical biosensing platform for the voltammetric monitoring of epinephrine

Epinephrine (EP) is one of the key neurotransmitter, which plays a vital role in the central nervous system. Current research report designates the development of biosensor based on the modification of glassy carbon electrode (GCE) with multi walled carbon nano tubes (MWCNTs) followed by drop casting of Tyrosinase (Ty) enzyme (Ty/MWCNTs/GCE) towards the sensitive monitoring of EP. The electrochemical behavior of EP at Ty/MWCNTs/GCE biosensor was examined and the redox mechanism was proposed. The developed Ty/MWCNTs/GCE was characterized by electrochemical techniques such as cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and tafel plot studies. The influence of pH of phosphate buffer solution (PBS) on the electrochemical redox behavior of EP was observed and pH-7.0 was identified as optimal pH value. The electrochemical kinetic parameters such as heterogeneous rate constant, diffusion coefficient and charge transfer coefficient values were evaluated. The limit of detection and limit of quantification values were evaluated. The low apparent Michaelis – Menten constant (K_m^app) was determined as 0.159 mM, demonstrating the immense catalytic activity of Ty enzyme. Repeatable, reproducible and stable nature of the fabricated Ty/MWCNTs/GCE was successfully examined. Finally, the developed biosensor was tested for the practical application in quantification of EP in human serum samples.     

DIFFUSION RESISTANCE IN LEAVES AS RELATED TO THEIR STOMATAL ANATOMY AND MICRO-STRUCTURE

Transpiration from a plant leaf depends upon the water vapor pressure gradient between the substomatal cavity and the free air beyond the leaf. Transpiration also depends inversely on the resistance of the diffusion pathway through the substomatal cavity, stomate, and surface boundary layer. The value of the diffusion resistance is derived mathematically for Zebrina pendula, Medicago sativa, and Pinus resinosa. The vapor pressure gradient depends on the leaf temperature and therefore is related to the energy budget of the leaf. The exact solution of the diffusion equation is described and limiting examples discussed. The so-called “diameter law” is a special case which is distinctly limited in its application.     

Label-free electrical detection of DNA hybridization for the example of influenza virus gene sequences

Microarrays based on DNA-DNA hybridization are potentially useful for detecting and subtyping viruses but require fluorescence labeling and imaging equipment. We investigated a label-free electrical detection system using electrochemical impedance spectroscopy that is able to detect hybridization of DNA target sequences derived from avian H5N1 influenza virus to gold surface-attached single-stranded DNA oligonucleotide probes. A 23-nt probe is able to detect a 120-nt base fragment of the influenza A hemagglutinin gene sequence. We describe a novel method of data analysis that is compatible with automatic measurement without operator input, contrary to curve fitting used in conventional electrochemical impedance spectroscopy (EIS) data analysis. A systematic investigation of the detection signal for various spacer molecules between the oligonucleotide probe and the gold surface revealed that the signal/background ratio improves as the length of the spacer increases, with a 12- to 18-atom spacer element being optimal. The optimal spacer molecule allows a detection limit between 30 and 100 fmol DNA with a macroscopic gold disc electrode of 1 mm radius. The dependence of the detection signal on the concentration of a 23-nt target follows a binding curve with an approximate 1:1 stoichiometry and a dissociation constant of KD=13+/-4 nM at 295 K.     

Molecular and structural modeling of the Phanerochaete flavido-alba extracellular laccase reveals its ferroxidase structure

The fungus Phanerochaete flavido-alba is highly efficient in the oxidation of olive oil wastewater-derived polyphenols. This capability is largely due to the action of a multicopper-oxidase (MCO), encoded by the pfaL gene. We describe the sequence and organization of pfaL gene and the biochemical characterization and predicted 3D structural model of the encoded protein. pfaL gene organization and peptide sequence are highly similar to those of P. chrysosporium MCOs. However, PfaL is the first MCO in the Phanerochaete genus to show evident laccase activity. Phylogenetic analysis places PfaL in a differentiated sub-branch of ferroxidases. Protein structure analysis reveals close similarity of PfaL and ferroxidases and provides clues about the differences of activity between both types of enzymes. To summarize, P. flavido-alba laccase is the first enzyme in the novel and biochemically poorly defined group of “ferroxidases/laccases” that shows efficacious oxidation of laccase substrates, biotechnologically exploitable in bioremediation approaches.     

Specific detection of Mycobacterium sp. genomic DNA using dual labeled gold nanoparticle based electrochemical biosensor

The present study was aimed at the development and evaluation of a DNA electrochemical biosensor for Mycobacterium sp. genomic DNA detection in a clinical specimen using a signal amplifier as dual-labeled AuNPs. The DNA electrochemical biosensors were fabricated using a sandwich detection strategy involving two kinds of DNA probes specific to Mycobacterium sp. genomic DNA. The probes of enzyme ALP and the detector probe both conjugated on the AuNPs and subsequently hybridized with target DNA immobilized in a SAM/ITO electrode followed by characterization with CV, EIS, and DPV analysis using the electroactive species para-nitrophenol generated by ALP through hydrolysis of para-nitrophenol phosphate. The effect of enhanced sensitivity was obtained due to the AuNPs carrying numerous ALPs per hybridization and a detection limit of 1.25 ng/ml genomic DNA was determined under optimized conditions. The dual-labeled AuNP-facilitated electrochemical sensor was also evaluated by clinical sputum samples, showing a higher sensitivity and specificity and the outcome was in agreement with the PCR analysis. In conclusion, the developed electrochemical sensor demonstrated unique sensitivity and specificity for both genomic DNA and sputum samples and can be employed as a regular diagnostics tool for Mycobacterium sp. monitoring in clinical samples.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-asparaginase in<Emphasis Type="Italic"> Enterobacter aerogenes</Emphasis> expressing<Emphasis Type="Italic"> Vitreoscilla</Emphasis> hemoglobin for efficient oxygen uptake

This study is the first utilizing Vitreoscilla hemoglobin in a heterologous bacterium, Enterobacter aerogenes, to determine the effect of such a highly efficient oxygen-uptake system on the production of l-asparaginase, an enzyme that has attracted considerable attention due to its anti-tumor activity. Here, we show that the Vitreoscilla hemoglobin expressing strain has from 10-fold to more than two orders of magnitude lower l-asparaginase activity than the wild type or the control without the Vitreoscilla hemoglobin gene under different aeration conditions. Aeration and agitation were also determining factors for enzyme production. The enzyme activity was reduced considerably under both full aerobic and anaerobic conditions, while the highest enzyme activity was determined in cultures under low aeration and low agitation. Also, the effect of different concentrations of glucose on enzyme production showed catabolic repression. Glucose at 1% caused almost total inhibition of enzyme activity, while at 0.1% it showed a slightly stimulatory effect on enzyme production, compared with glucose-free medium.