Biosensor for asparagine using a thermostable recombinant asparaginase from Archaeoglobus fulgidus

Asparaginase from the hyperthermophilic microorganism Archaeoglobus fulgidus was cloned and expressed in Escherichia coli as a fusion protein with a polyhistidine tail. After heat treatment to denature most of the native E. coli proteins, the enzyme was purified by an immobilized metal ion affinity chromatography method. The activity of the enzyme was determined by monitoring the change in ammonium concentration in solution. It was found that the enzyme is thermostable at temperatures as high as 85 degrees C. The KM for L-asparagine was 8 x 10(-5) and 5 x 10(-6) M at 37 and 70 degrees C, respectively. The catalytic activity for L-asparagine was 5-fold higher than for D-asparagine. The enzyme was immobilized in front of an ammonium-selective electrode and used to develop a biosensor for asparagine. The biosensor had a detection limit of 6 x 10(-5) M for L-asparagine. Unlike a sensor based on asparaginase from E. coli, the biosensor based on recombinant asparaginase from A. fulgidus demonstrated higher stability.     

Operation of a miniature redox hydrogel-based pyruvate sensor in undiluted deoxygenated calf serum

An amperometric sensor for the detection of pyruvate in biological fluids was formed by modifying the tip of a 0.25 mm gold wire with a layer of electrically "wired" recombinant pyruvate oxidase (POP). The sensor did not require O2 for its operation. The electroactive area of the tip of the microwire was increased by electrodeposition of platinum black. The POP was adsorbed on the platinum black and then "wired" with the cross-linked, subsequently deposited poly(4-vinylpyridine), part of the pyridine functions of which were complexed with [Os(bpy)2Cl](+/2+) and part quaternized with 2-bromoethylamine. In the resulting thin layer the POP was well "wired". When the electrode was poised at 0.4 V vs Ag/ AgCl, the sensitivity at pH 6 was 0.26 A cm(-2) M(-1) and the current increased linearly with the pyruvate concentration through the 2 x 10(-6) - 6 x 10(-4) M range. Thiamine diphosphate, flavin adenine dinucleotide, and MgCl2 were not required for the assay, but stabilized the stored enzyme electrode. Placement of a dialysis membrane (MWCO 3500) on the electrode alleviated the severe interference of ascorbate. In calf serum, the detection limit was 30 microM, suggesting that the electrode might be used in the continuous monitoring of pyruvate in hypoxic organs.     

Electrochemical NO2 sensor using a NiFe1.9Al0.1O4 oxide spinel electrode

A novel solid-state electrochemical sensor using (Sc2O3)0.08(ZrO2)0.92 (ScSZ) electrolyte solid and a NiFe1.9Al0.1O4 oxide spinel electrode was tested for the detection of NO2 at temperatures greater than 700 degrees C for automobile applications. The sensor was found to respond rapidly, reproducibly, and selectively to NO2 at 703 and 740 degrees C. The response time of the sensor was approximately 8 s, and the recovery time was 10 s at both 703 and 741 degrees C. The response of the sensor was highly reproducible to the change in concentration of NO2 and also showed negligible cross-sensitivity to potentially interfering gases such as O2, CO, and CH4 in the gas stream.     

GaPO4 sensors for gravimetric monitoring during atomic layer deposition at high temperatures

The quartz crystal microbalance is extremely useful for in situ monitoring of thin-film growth by atomic layer deposition (ALD) in a viscous flow environment. Unfortunately, conventional AT-quartz sensors are limited to growth temperatures below approximately 300 degrees C. Gallium orthophosphate (GaPO4) is an alternative piezoelectric material offering much greater high-temperature frequency stability than AT-quartz (SiO2). Our measurements reveal that the temperature coefficient for Y-11 degrees GaPO4 decreases linearly with temperature reaching 3 Hz/ degrees C at 450 degrees C. In contrast, the temperature coefficient for the SiO2 sensor increases as the cube of the sensor temperature to 650 Hz/ degrees C at 390 degrees C. To examine the effect of temperature fluctuations on the sensor frequency, we exposed the SiO2 and GaPO4 sensors to helium pulses at 400 degrees C. The resulting frequency change measured for the SiO2 sensor was approximately 40 times greater than that of the GaPO4 sensor. Next, we performed Al2O3 ALD using alternating tri-methylaluminum/water exposures at 400 degrees C and monitored the growth using the SiO2 and GaPO4 sensors. The GaPO4 sensor yielded well-defined pulse shapes in agreement with predictions, while the SiO2 pulses were severely distorted. Measurements during TiO2 ALD using alternating titanium tetrachloride/water exposures at 450 degrees C with the GaPO4 sensor also showed well-defined ALD mass steps.     

Temperature alternation by an on-chip microheater to reveal enzymatic activity of beta-galactosidase at high temperatures

A method to measure enzymatic activity at high temperatures by rapid temperature alternation of a microreactor with a microheater is proposed. On-chip microreactor and microheater were integrated on a glass plate by MEMS technology; this microheater can control the temperature of the microreactor with a response speed of 34.2 and 31.5 K/s for temperature rise and fall, respectively, with an accuracy of 3 degrees C. The enzyme, beta-galactosidase, was revealed to survive short exposure (4-s pulses) to temperatures above that which would "normally" denature them. Its activity at 60 degrees C was revealed to be approximately 4 times greater than that at room temperature. This method not only gives new kinetic information in biochemistry but also enables application in highly sensitive biosensors.     

Short- and long-term stability of resonant quartz temperature sensors

A new miniaturized design of the thermosensitive quartz resonator (TSQR) using an NLC cut (yxl/ -31 degrees 30') with a fundamental frequency of 29.3 MHz was created in the Acoustoelectronics Laboratory of ISSPBAS for use in a wide temperature range (4.2 K to 450 K) as highly sensitive quartz temperature sensors (QTS). This paper presents the results of the investigations of the short- and long-term frequency stability of QTS. The short-term frequency stability of QTS was measured for averaging times up to 150 s at three constant temperatures: liquid helium (4.2 K), liquid nitrogen (77 K), and melting ice (0 degrees C). The short-term frequency stability is 6.8 * 10(-9) at 0 degrees C for t = 15 s, which permits a temperature sensitivity of 2 * 10(-4) K. The long-term stability (aging) was investigated at room temperature and at 80 degrees C for 500 days. The aging characteristics at 25 degrees C and 80 degrees C are compared. It was observed that the frequency change does not exceed 5 * 10(-7) after the 25th day of accelerated aging at 80 degrees C. This guarantees a reliable operation of the sensor, without additional calibration, for several years.     

Hydrogen sensor based on Au and YSZ/HgO/Hg electrode for in situ measurement of dissolved H2 in high-temperature and -pressure fluids

Gold as a hydrogen-sensing electrode for in situ measurement of dissolved H2 in aqueous solutions under extreme conditions is reported. The dissolved H2 sensor, constructed with a Au-based sensing element and coupled with a YSZ/HgO/Hg electrode, is well suited for determining dissolved H2 concentrations of aqueous fluids at elevated temperatures and pressures. The Au electrode is made of Au wire mounted in a quartz bar, which can be pressurized and heated in the high-pressure and -temperature conditions. The Au-YSZ sensor has been tested for its potential response to the concentrations of dissolved H2 in fluids by using a flow-through reactor at high temperatures up to 400 degrees C and pressures to 38 MPa. Good sensitivity and linear response between the hydrogen concentrations in the fluids and the H2 sensor potentials are reported for hydrogen gas in the concentration range of 0.1-0.001 M H2 in aqueous fluids at temperatures up to 340 degrees C and 30 MPa. Nernstian response of the cell potential to dissolved H2 in fluids was determined at 340 degrees C and 30 MPa, described as follows: DeltaE = 0.9444 + 0. 0603 log m H2 The experimental results indicate that the Au-YSZ/HgO/Hg cell can be used to measure the solubility of H2 in aqueous fluid at temperatures and pressures near to the critical state of water. Thus, this type of Au hydrogen sensor could be easily used for in situ measurement of H2 in hydrothermal fluids in a high-pressure vessel, or at midocean ridge, due to its structure of compression resistance.     

Employing the metabolic "branch point effect" to generate an all-or-none, digital-like response in enzymatic outputs and enzyme-based sensors

Here, we demonstrate a strategy to convert the graded Michaelis-Menten response typical of unregulated enzymes into a sharp, effectively all-or-none response. We do so using an approach analogous to the "branch point effect", a mechanism observed in naturally occurring metabolic networks in which two or more enzymes compete for the same substrate. As a model system, we used the enzymatic reaction of glucose oxidase (GOx) and coupled it to a second, nonsignaling reaction catalyzed by the higher affinity enzyme hexokinase (HK) such that, at low substrate concentrations, the second enzyme outcompetes the first, turning off the latter's response. Above an arbitrarily selected "threshold" substrate concentration, the nonsignaling HK enzyme saturates leading to a "sudden" activation of the first signaling GOx enzyme and a far steeper dose-response curve than that observed for simple Michaelis-Menten kinetics. Using the well-known GOx-based amperometric glucose sensor to validate our strategy, we have steepen the normally graded response of this enzymatic sensor into a discrete yes/no output similar to that of a multimeric cooperative enzyme with a Hill coefficient above 13. We have also shown that, by controlling the HK reaction we can precisely tune the threshold target concentration at which we observe the enzyme output. Finally, we demonstrate the utility of this strategy for achieving effective noise attenuation in enzyme logic gates. In addition to supporting the development of biosensors with digital-like output, we envisage that the use of all-or-none enzymatic responses will also improve our ability to engineer efficient enzyme-based catalysis reactions in synthetic biology applications.     

Short- and long-term effects of temperature on the Anammox process

The application of the Anammox process has been usually focused on the treatment of wastewater with temperatures around 30 degrees C in order to operate under optimum conditions. In this work, the feasibility of the application of the Anammox process at lower temperatures has been tested. First, the short-term effects of temperature on the Anammox biomass were studied using batch tests. An activation energy of 63 kJ mol(-1) was calculated and the maximum activity was found at 35-40 degrees C. Activity tests done at 45 degrees C showed an irreversible loss of the activity due to the biomass lysis. A SBR was operated at different temperatures (from 30 to 15 degrees C) to determine the long-term effects. The system was successfully operated at 18 degrees C but when temperature was decreased to 15 degrees C, nitrite started to accumulate and the system lost its stability. Adaptation of biomass to low temperatures was observed when the specific activities obtained during first batch tests are compared to those obtained during the operation of the SBR.     

Cyanide detection using a substrate-regenerating, peroxidase-based biosensor

An enzyme-based, dual working electrode system is described for the sensing of cyanide. Horseradish peroxidase (HRP) is incorporated as the sensing element. A continuous monitoring of oxidative activity by the enzyme results through the generation and regeneration of substrates at the electrode surfaces. Thus, HRP is oxidized by hydrogen peroxide generated from dissolved oxygen, at the primary electrode, and then reduced through the secondary electrode by mediated electron transfer using ferrocene as a carrier. Ferrocene regeneration at this electrode is proportional to the intrinsic activity of HRP. The dynamics of the system are investigated by using a rotating ring-disk electrode. The enzyme is immobilized to provide better control over its catalytic activity and to increase the lifetime of the biosensor. Cyanide inhibition of current can be modeled by reversible binding kinetics. Detection of cyanide is possible in submicromolar (ppb) concentrations, with a half maximal response at 2 microM. The response time for detection of introduced cyanide is within 1 s. The sensor can be operated between 5 and 40 degrees C, and cyanide inhibition is unaffected by pH changes between 5 and 8. The sensor is reproducible for cyanide determination and is stable for over 6 months.     

Study of the effect of some organic solvents on the activity and stability of glucose oxidase

The relative activity and thermal stability of native and covalently immobilized glucose oxidase (GOD) onto polyamide-6 (PA-6) membrane was studied at temperatures of 28, 45 and 60 °C for 10 h and in the presence of organic solvents (methylol, ethyleneglycol, glycerol) with concentrations 10%, 30% and 60%. It was proven that immobilized GOD had better stability than the native one in all the three organic solvents. At 28 °C, the strongest activating and stabilizing effect on the free GOD was observed with 10% methylol and on the immobilized GOD with 10% and 30% methylol. The addition of certain concentrations of ethyleneglycol and glycerol to the enzyme at higher temperatures was found to stabilize the enzyme molecule. At temperatures of 45 and 60 °C, the strongest stabilizing effect on both forms of the enzyme was exerted by glycerol (optimal stabilizing concentration of 10%). It was concluded that the most stable form of the enzyme was GOD covalently immobilized onto PA-6 membrane in the presence of 10% solution of glycerol.     

Producing "self-plasticizing" ion-selective membranes

Polymer membranes have been explored for the analysis of ions that do not require plasticizers and with photocurable properties. This work was focused on investigating the viability of the methacrylic-acrylic copolymers as new self-plasticizing membrane matrixes for ion-selective electrodes or other ion-sensor applications. Copolymers with glass transition temperatures ranging from -20 to -44 degrees C could be prepared without added plasticizer and were found to be functional as ion-selective membranes. Both free-radical solution polymerization and photopolymerization could be used, and "self-plasticizing" behavior of copolymers was observed with a high alkylacrylate (R = C4) content. This was found to be compatible with most commercially available ionophores, and sensors for potassium, sodium, calcium and pH were fabricated entirely by photocure procedures; single-step procedures for the immobilization of benzo-15-crown-5 ionophore on these self-plasticizing copolymer matrixes were also developed. Even though the ionophore was immobilized, potentiometric studies revealed that the ionophore remained functional, and thus, these copolymers have the advantage of suffering neither leaching of ionophore nor plasticizers. All these sensors exhibited a Nernstian or near Nernstian response with selectivity comparable to plasticized PVC membranes or other plasticized and photocurable polymer membranes. The long-term response of the potassium sensor with immobilized ionophore and the sodium sensor showed little deterioration for as long as one month and three months, respectively, under continuous use.     

Erbium-doped optical fiber fluorescence temperature sensor with enhanced sensitivity,a high signal-to-noise ratio,and a power ratio in the 520-530- and 550-560-nm bands

We analyze and predict the performance of a fiber-optic temperature sensor from the measured fluorescence spectrum to optimize its design. We apply this analysis to an erbium-doped silica fiber by employing the power-ratio technique. We develop expressions for the signal-to-noise ratio in a band to optimize sensor performance in each spectral channel. We improve the signal-to-noise ratio by a factor of 5 for each channel, compared with earlier results. We evaluate the analytical expression for the sensor sensitivity and predict it to be approximately 0.02 degrees C(-1) for the temperature interval from room temperature to above 200 degrees C, increasing from 0.01 degrees C(-1) at the edges of the interval to 0.03 degrees C(-1) at the center, at 100-130 degrees C. The sensitivity again increases at temperatures higher than 300 degrees C, delineating its useful temperature intervals.     

Fiber-optic voltage sensor based on a Bi12TiO20 crystal

A fiber-optic voltage sensor based on the longitudinal Pockels effect in a Bi(12)TiO(20) crystal is described. The use of a special backreflecting prism as a phase-retarding element is shown to improve the sensitivity and temperature stability of the sensor. A comparison between the temperature properties of the glass backreflecting prism and that of a quarter-wave plate is derived. The sensor demonstrates temperature stability of +/-1.5% from -20 degrees C to 60 degrees C and sensitivity of 0.145% per 1 V(rms) at 850 nm without the use of an additional temperature control channel.     

Ultra-high oxidation resistance of suspended single-wall carbon nanotube bundles grown by an "all-laser" process

Single-wall carbon nanotubes (SWCNTs) were laterally grown on SiO2/Si substrates by means of an "all-laser" growth process. Our "all-laser" process stands out by its exclusive use of the same pulsed UV laser, first, to deposit the CoNi nanocatalyst and, second, to grow SWCNTs through the laser ablation of a pure graphite target. The "all-laser" grown SWCNTs generally self-assemble into bundles (5-15 nm-diam.) sprouting from the CoNi nanocatalyst and laterally bridging the 2 microm gap separating adjacent catalysed electrodes (in either "suspended" or "on-substrate" geometries). A comparative study of the oxidation resistance of both suspended and on-substrate SWCNTs was achieved. The "all-laser" grown SWCNTs were subjected to annealing under flowing oxygen at temperatures ranging from 200 to 1100 degrees C. Systematic scanning electron microscopy observations combined with micro-Raman analyses revealed that more than 20% of suspended nanotubes were still stable at temperatures as high as 900 degrees C under flowing O2 while the on-substrate counterpart were completely burnt out at this temperature. Accordingly, the activation energy, as deduced from the Arrhenius plot, of the suspended SWCNTs is found to be as high as approximately 180 kJ mol(-1) (approximately 9 times higher than that of the on-substrate ones). The high quality (almost defect-free) of the nanotubes synthesized by the "all-laser" approach, their protected tips into the embedded CoNi catalyst nanolayer together with their suspended geometry are thought to be responsible for their unprecedented ultra-high oxidation resistance. This opens up new prospects for the use of these suspended nanotubes into nanodevices that have to operate under highly oxidizing environments.     

Thermal and mechanical enhancements of polyetherimide/multi-walled carbon nanotube composite performance using "Solid Nano-Nectar" assisted melt dispersion

Manufacturing industrial-volume composites with well controlled uniform dispersion can be challenging, and is especially true for high performance polymers, such as polyetherimide (PEI). Toward that end, we report here a simple and versatile 2-Step processing approach which is based on formation of a high concentration of multi-walled carbon nanotube (MWCNT) filled PEI, which we call "Solid Nano-Nectar" (SNN). In this study we found surprisingly strong influences of the solvents that were used in the SNN preparation for dispersion of MWCNTs on the properties of the composites. The thermal conductivity of the SNN (made using DMF solvent) incorporated composites was about 72% higher than pure PEI, and 50% higher than that from 1-Step processing at same loading of 0.5 wt%. The thermal stability increased by 13 degrees C from this composite prepared by 1-Step processing, but a more noticeable change in thermal stability of 33 degrees C was observed at same loading using the 2-Step process. Such improvement was also observed in dynamic mechanical properties.     

Multimicrobial sensor using microstructured three-dimensional electrodes based on silicon technology

Two microbial strains with different substrate spectra were immobilized separately within a single biosensor chip featuring four individually addressable platinum electrodes. These were sputtered onto the inner surface of four isolated pyramidal cavities ("containments") micromachined on a silicon wafer. The biosensor chip was integrated into a flow-through system to measure the oxygen consumption of the immobilized microorganisms in the presence of assimilable analytes. As a model system, a yeast for the determination of biochemical oxygen demand (BOD) and a strain capable of degrading polycyclic aromatic hydrocarbons (PAH) were chosen. It was shown that the simple and mass-producible containment sensor exhibits good performance data: lower detection limit 0.1 mg/L naphthalene and 1 mg/L sensor-BOD; calibration range up to 30 mg/L; precision 3-6%; response time 2-3 min; service life up to 40 days; shelf life at 4 degrees C 6 months. The versatility of the multimicrobial sensor was demonstrated by measuring ordinary municipal wastewater samples as well as various aqueous samples contaminated with PAH. The concept of a multimicrobial sensor not only enlarges the substrate spectrum for sum parameters such as BOD but leads to additional information which allows for a more differentiated and immediate knowledge of sample composition. Using chemometrical data analysis, the multimicrobial sensor lays a foundation for developing an "electronic tongue".     

Temperature-insensitive fiber Bragg grating tilt sensor

A temperature-insensitive optical fiber tilt sensor is presented. The sensor scheme uses a prestrained fiber Bragg grating to sense the strain, which depends on the tilt angle. To compensate for the temperature effect, materials that have different linear thermal expansion behaviors are used for implementation of the sensor body. The differentiation in the linear thermal expansion would then cause a counter effect to the original temperature effect. Experimental tests show an accuracy of +/-0.167 degrees in tilt angle measurement. A temperature stability better than +/-0.33 degrees over the temperature range from 27 degrees C to 75 degrees C is demonstrated. The resolution 0.0067 degrees in tilt angle measurement is achieved by using our preliminary sensor with a dimension of 1 6 x 5 x 5 cm(3).     

Long-term stability and performance characteristics of crystal quartz gauge at high pressures and temperatures

Good long-term stability of high precision quartz pressure sensors is necessary for various applications ranging from pressure transient analysis to permanent monitoring systems for optimal reservoir management in the petroleum industry. A crystal quartz gauge (CQG(1 )) is a dual-mode, thickness-shear, quartz pressure sensor that has been used in oil field services for the past 8 years. High accuracy, resolution, and fast response time of this sensor enable a reliable estimate of formation permeability and oil/water interfaces in reservoirs that help reduce the overall cost of oil and gas production. The sensing resonator characteristics can be described in terms of equivalent circuit parameters (motional resistance and capacitance), resonator-Q and the short-term frequency stability of both the B- and C-modes of vibrations at various temperatures. The pressure reading errors of manufactured gauges are less than 8.89 kPa (1 psi) (plus 0.01% of the reading because of the uncertainty of the dead-weight tester). The pressure resolution is better than 20.7 Pa (0.003 psi) over a 1-s gate time. An extremely effective dynamic compensation algorithm yields corrected pressure readings with a very fast response time as short as a strain-gauge-based pressure transducer while retaining the high performance of a quartz gauge. Recent long-term stability tests of CBGs show a negligibly small drift of the order of a few tenths of 1 psi (0.1 psi=689 Pa) at 103 MPa (15 kpsi) and 175 degrees C for a period of more than 1 yr. These results confirm that the CQG characteristics exceed the demanding specifications for both the well tests and permanent monitoring systems.     

Dual-enzyme fiber-optic biosensor for glutamate based on reduced nicotinamide adenine dinucleotide luminescence.

Response characteristics are presented for a dual-enzyme fiber-optic biosensor for glutamate. An enzyme layer composed of glutamate dehydrogenase (GDH) and glutamate-pyruvate transaminase (GPT) is used to produce reduced nicotinamide adenine dinucleotide (NADH) at the tip of a fiber-optic probe. NADH luminescence is monitored through this probe and the measured fluorescence intensity is related to the concentration of glutamate. GDH catalyzes the formation of NADH, and GPT drives the GDH reaction by removing a reaction product and regenerating glutamate. Optimal response is obtained in a pH 7.4 Tris-HCl buffer maintained at 25 degrees C in the presence of 4 mM NAD+ and 10 mM L-alanine. The temperature profile reveals a strong negative temperature effect which is attributed to the temperature dependency of NADH luminescence. Under optimal conditions, the sensor sensitivity is 0.127 nA/microM over the 1-10 microM concentration range, the detection limit is 0.13 microM, and response times range from 4 to 8 min. The sensor response is stable for 12 days when stored at 4 degrees C. Selectivity for glutamate is excellent over most of the common amino acids as well as ascorbic acid, uric acid, taurine, and GABA. Only slight responses were observed for glutamine and lysine. The effect of ammonia on the glutamate response was found to be minimal at total ammonia nitrogen concentrations as high as 200 microM.