Functional Expression of a Mo-Cu-Dependent Carbon Monoxide Dehydrogenase (CODH) and Its Use as a Dissolved CO Bio-microsensor.

Herein, we report the heterologous expression in Escherichia coli of a Mo-Cu-containing carbon monoxide dehydrogenase (Mo-Cu CODH) from Hydrogenophaga pseudoflava, which resulted in an active protein catalyzing CO oxidation to CO2. By supplying the E. coli growth medium with Na2MoO4 (Mo) and CuSO4 (Cu), the Mo-Cu CODH metal cofactors precursors, the expressed L-subunit was found to have CO-oxidation activity even without the M- and S- subunits. This successful expression of CO-oxidizing-capable single L-subunit provides direct evidence of its role as the catalytic center of Mo-Cu CODH that has not been discovered and studied before. Subsequently, we used the expressed protein to construct a CO bio-microsensor based on a newly developed fast and sensitive Clark-type CO2 transducer using an aprotic solvent/ionic liquid electrolyte. The CO bio-microsensor exhibited a linear response to CO concentration in the 0-9 µM range, with a limit of detection (LOD) of 15 nM CO. The sensor uses a mixture of Mo-Cu CODH's L-subunit/Mo, Cu cofactors/methylene blue, confined in the enzyme chamber that is placed in front of a CO2 transducer. The optimized sensor's sensitivity and performance were retained to levels of at least 80% for 1 week of continuous polarization and operation in an aqueous medium. We have also demonstrated the use of an alkaline front-trap solution to make a completely O2/CO2 interference-free microsensor. The CO bio-microsensor developed in this study is potentially useful as an analytical tool for the detection of trace CO in dissolved form for monitoring dissolved CO concentration dynamics in natural or synthetic systems.

Urea Biosensor Based on a CO2 Microsensor.

Urea sensors based on electrodes in direct contact with the medium have limited long-term stability when exposed to complex media. Here, we present a urea biosensor based on urease immobilized in an alginate polymer, buffered at pH 6, and placed in front of a newly developed fast and sensitive CO2 microsensor, where the electrodes are shielded by a gas-permeable membrane. The CO2 produced by the urease in the presence of urea diffuses into the microsensor and is reduced at a Ag cathode. Oxygen interference is prevented by a Cr2+ trap. The 95% response time to changes in urea concentration was 120 s with a linear calibration curve in the range 0-1000 µM and a detection limit of 1 µM. The Ni2+ cofactor to improve sensor performance was continuously supplied from a reservoir behind the sensor tip. The stability of the urea sensor was optimized by the addition of bovine serum albumin as a stabilizer to the urease/alginate mixture that was cross-linked with glutaraldehyde and Ca2+ ions. This immobilization strategy resulted in about 70% of the initial urea sensor sensitivity after two weeks of continuous operation. The sensor was successfully tested in blood serum.

Ion Selective Amperometric Biosensors for Environmental Analysis of Nitrate, Nitrite and Sulfate.

Inorganic ions that can be redox-transformed by living cells can be sensed by biosensors, where the redox transformation gives rise to a current in a measuring circuit. Such biosensors may be based on enzymes, or they may be based on application of whole cells. In this review focus will be on biosensors for the environmentally important ions NO3-, NO2-, and SO42-, and for comparison alternative sensor-based detection will also be mentioned. The developed biosensors are generally characterized by a high degree of specificity, but unfortunately also by relatively short lifetimes. There are several investigations where biosensor measurement of NO3- and NO2- have given new insight into the functioning of nitrogen transformations in man-made and natural environments such as sediments and biofilms, but the biosensors have not become routine tools. Future modifications resulting in better long-term stability may enable such general use.

Fast Responding Amperometric CO2 Microsensor with Ionic Liquid-Aprotic Solvent Electrolytes.

Knowledge about the microscale distribution of CO2 is essential in many environmental and technical settings, and electrochemical CO2 sensing may be optimized to yield such information. The performance of a Clark-type CO2 sensor was greatly improved by adding 20% dimethylformamide (DMF) to the ionic liquid 1-ethyl-3-methylimidazolium dicyanamide (EMIM-DCA) previously used as an electrolyte. The addition of DMF resulted in a much faster response to increasing (95% response of about 100 s) or decreasing CO2 concentration, a negligible interference from low concentrations of N2O, and a signal temperature dependence similar to that of O2 microsensors. The use of 80% EMIM-DCA/20% DMF as an electrolyte leads to CO2 reduction at -0.72 V (vs standard hydrogen electrode), reducing the overpotential by 0.2 V as compared to the use of 100% EMIM-DCA. The CO2 microsensor has a calculated limit of detection of 0.5 Pa CO2, and sensors optimized for high sensitivity exhibited a linear response within the range of 0-4.6 kPa (0-1.7 mM) CO2. A set of four sensors exhibited no noticeable change of zero current and CO2 sensitivity during 4 months of continuous polarization.

Controlling Voltage Reversal in Microbial Fuel Cells.

Microbial fuel cell (MFC) systems have been developed for potential use as power sources, along with several other applications, with bacteria as the prime factor enabling electrocatalytic activity. Limited voltage and current production from unit cells limit their practical applicability, so stacking multiple MFCs has been proposed as a way to increase power production. Special attention is paid to voltage reversal (VR), a common occurrence in stacked MFCs, and to identifying the mechanisms underlying this phenomenon. We also proposed realistic perspectives on stacked MFCs in an effort to control and suppress VR by balancing the kinetics in the system, such as using enriched electroactive microorganisms or altering the circuitry mode.

Electrochemical Enzyme-Linked Sandwich Assay with a Cellulase Label for Ultrasensitive Analysis of Synthetic DNA and Cell-Isolated RNA.

Electrochemical enzyme-linked sandwich assays on magnetic beads (MBs) refer to one of the most sensitive approaches for analysis of nonamplified nucleic acid samples, with redox enzymes being routinely used as labels. Here, we report a sensitive and inexpensive electrochemical nucleic acid sandwich assay on MBs that exploits a hydrolytic enzyme cellulase as a label, while MBs are used for preconcentration and bioseparation of analyzed samples. Binding of target DNA or RNA to capture DNA-modified MB triggers sandwich assembly and its labeling with cellulase. Application of the assembled sandwich to the electrodes covered with insulating nitrocellulose films induces film digestion by the cellulase label and pronounced changes in the electrical properties of the electrodes, the extent of the changes being proportional to the concentration of the analyzed nucleic acids. Down to 1 amol of Lactobacillus brevis specific synthetic DNA and rRNA isolated from L. brevis cells could be detected in 1 mL samples in the overall from 2 to 3 h assay. The assay is universal and can be adapted for point-of-care-testing and for in-field environmental and microbiomic analysis of unamplified samples of any DNA/RNA sequences.

Electrochemical Assay for a Total Cellulase Activity with Improved Sensitivity.

Electrochemical methods allow fast and inexpensive analysis of enzymatic activities. Here, we report a simple and yet efficient electrochemical assay for the total activity of cellulase, a hydrolytic enzyme widely used in food and textiles industries, and for production of bioethanol. The assay exploits the difference in electrochemical signals from a soluble redox indicator, ferricyanide, on nitrocellulose films treated by cellulases. Ferricyanide electrochemistry is totally inhibited on graphite electrodes modified with an insulating nitrocellulose film and is evoked after the cellulase treatment. Ferricyanide voltammetric responses correlate with the increased permeability of the films and electrochemically active surface area of electrodes becoming accessible to the ferricyanide reaction after nitrocellulose digestion by cellulase. Trichoderma and Aspergillus niger cellulases activities were determined in a 5 min assay with a sensitivity of 10-8 U per assay, being 103-104-fold more sensitive than the standard commercially available optical assays. That makes the developed electrochemical approach the most prospective cost-effective alternative both for research and automated industrial applications.

Graphitized-Carbon-Nanofiber Paper-Enzyme Electrode Fabrication Through Non-Covalent Modification for Enzyme Biofuel Cell Application.

Carbon nanofibers are an emerging smart material that are promising for use as a biosensor and a biofuel cell transducer material due to their morphological and electrochemical characteristics. In particular, graphitized carbon nanofibers possess unique structures of graphite-like edges within their high surface area that provide a large active site for enzyme attachment. For a specific application such as a biofuel cell, which requires highly stable electrical communication and electricity generation, non-covalent enzyme immobilization using bifunctional molecule is suggested as an appropriate approach because it does not change the carbon hybridization from sp2 to sp3 as covalent immobilization by acid treatment does. Graphitized carbon-nanofiber paper (GCNFp) electrode were fabricated through dispersion-filtration method in which glucose oxidase as model enzyme were immobilized by a bifunctional molecule that forms π-π stacking of the pyrene moiety with the nanofiber wall coupled by a reactive end-amine reaction. This system provides a practical enzyme-electrode hybrid that facilitates comparatively faster enzyme-electrode electrical communication than other system using similar material, as calculated from the heterogeneous electron-transfer rate constant (K(s)) which was 5.45 s(-1).

Functionalized Polyacrylonitrile Nanofibrous Membranes for Covalent Immobilization of Glucose Oxidase.

Nanofibrous membrane (NFM) with uniform morphology and large surface area was prepared from 10% solution of polyacrylonitrile (PAN) in N,N-dimethylformamide by electrospinning technique. NFM was chemically modified for use as a support for the immobilization of glucose oxidase. Chemical modification of NFM was carried out by two different methods. In the first method, the cyano groups of PAN were modified to amino groups by a two-step process, while in the second method the carboxylic groups were generated first and then further reacted with hexamethylene diamine to create a reactive spacer arm for the immobilization of enzyme. Scanning electron microscopy studies showed that the surface morphology of NFM was not changed by chemical modification and its mechanical strength was improved. The immobilized glucose oxidase (GOx) retained 54 and 60% of its original activity up to 25 cycles with the PAN NFMs modified by the first and the second method, respectively. The GOx-immobilized NFM from the second method showed promising performance with higher enzyme immobilization, activity retention, and favorable kinetic parameters.

Bioelectronic platforms for optimal bio-anode of bio-electrochemical systems: From nano- to macro scopes.

The current trend of bio-electrochemical systems is to improve strategies related to their applicability and potential for scaling-up. To date, literature has suggested strategies, but the proposal of correlations between each research field remains insufficient. This review paper provides a correlation based on platform techniques, referred to as bio-electronics platforms (BEPs). These BEPs consist of three platforms divided by scope scale: nano-, micro-, and macro-BEPs. In the nano-BEP, several types of electron transfer mechanisms used by electrochemically active bacteria are discussed. In the micro-BEP, factors affecting the formation of conductive biofilms and transport of electrons in the conductive biofilm are investigated. In the macro-BEP, electrodes and separators in bio-anode are debated in terms of real applications, and a scale-up strategy is discussed. Overall, the challenges of each BEP are highlighted, and potential solutions are suggested. In addition, future research directions are provided and research ideas proposed to develop research interest.

Gated electron transfer reactions of truncated hemoglobin from Bacillus subtilis differently orientated on SAM-modified electrodes.

Electron transfer (ET) reactions of truncated hemoglobin from Bacillus subtilis (trHb-Bs) are suggested to be implicated in biological redox signalling and actuating processes that may be used in artificial environment-sensing bioelectronic devices. Here, kinetics of ET in trHb-Bs covalently attached via its surface amino acid residues either to COOH- or NH2-terminated (CH2)2-16 alkanethiol SAM assembled on gold are shown to depend on the alkanethiol length and functionalization, not being limited by electron tunnelling through the SAMs but gated by ET preceding reactions due to conformational changes in the heme active site/at the interface. ET gating was sensitive to the properties of SAMs that trHb-Bs interacted with. The ET rate constant ks for a 1e(-)/H(+) reaction between the SAM-modified electrode and heme of trHb-Bs was 789 and 110 s(-1) after extrapolation to a zero length SAM, while the formal redox potential shifted 142 and 31 mV, for NH2- and COOH-terminated SAMs, respectively. Such domain-specific sensitivity and responsivity of redox reactions in trHb-Bs may be of immediate biological relevance and suggest the existence of bioelectronic regulative mechanisms of ET proceeding in vivo at the protein-protein charged interfaces that modulate the protein reactivity in biological redox signalling and actuating events.

High performance enzyme fuel cells using a genetically expressed FAD-dependent glucose dehydrogenase α-subunit of Burkholderia cepacia immobilized in a carbon nanotube electrode for low glucose conditions.

FAD-dependent glucose dehydrogenase (FAD-GDH) of Burkholderia cepacia was successfully expressed in Escherichia coli and subsequently purified in order to use it as an anode catalyst for enzyme fuel cells. The purified enzyme has a low Km value (high affinity) towards glucose, which is 463.8 µM, up to 2-fold exponential range lower compared to glucose oxidase. The heterogeneous electron transfer coefficient (Ks) of FAD-GDH-menadione on a glassy carbon electrode was 10.73 s(-1), which is 3-fold higher than that of GOX-menadione, 3.68 s(-1). FAD-GDH was able to maintain its native glucose affinity during immobilization in the carbon nanotube and operation of enzyme fuel cells. FAD-GDH-menadione showed 3-fold higher power density, 799.4 ± 51.44 µW cm(-2), than the GOX-menadione system, 308.03 ± 17.93 µW cm(-2), under low glucose concentration, 5 mM, which is the concentration in normal physiological fluid.

Prevalence of Escherichia coli in surface waters of Southeast Asian cities.

Surface water samples were collected from rivers which fed into large urban areas within Vietnam, Indonesia, Cambodia, and Thailand and were processed to enumerate Escherichia coli. Selected isolates were further characterized using PCR to detect the presence of specific virulence genes. Analyzing the four countries together, the approximate mean cfu/100 ml for E. coli counts in the dry season were log 4.3, while counts in the wet season were log 2.8. Of the 564 E. coli isolates screened for the presence of pathogenic genes, 3.9 % possessed at least one virulence gene. The most common pathogenic types found were Shiga toxin-producing E. coli isolates. These results reinforce the importance of monitoring urban surface waters for fecal contamination, that E. coli in these water environments may serve as opportunistic pathogens, and may help in determining the impact water usage from these rivers have on the public health of urban populations in Southeast Asia.