WO2006101963A1 - Detecteur d'hydrogene utilisant une reaction catalysee par des enzymes - Google Patents

Detecteur d'hydrogene utilisant une reaction catalysee par des enzymes Download PDF

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WO2006101963A1
WO2006101963A1 PCT/US2006/009495 US2006009495W WO2006101963A1 WO 2006101963 A1 WO2006101963 A1 WO 2006101963A1 US 2006009495 W US2006009495 W US 2006009495W WO 2006101963 A1 WO2006101963 A1 WO 2006101963A1
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solution
sensor
electron acceptor
concentration
hydrogen
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PCT/US2006/009495
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English (en)
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Zhonghui H. Fan
Brent J. Lutz
Baerbel Friedrich
Tanja Burgdorf
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University Of Florida Research Foundation, Inc.
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Publication of WO2006101963A1 publication Critical patent/WO2006101963A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/0004—Gaseous mixtures, e.g. polluted air
    • G01N33/0009—General constructional details of gas analysers, e.g. portable test equipment
    • G01N33/0027—General constructional details of gas analysers, e.g. portable test equipment concerning the detector
    • G01N33/0036—General constructional details of gas analysers, e.g. portable test equipment concerning the detector specially adapted to detect a particular component
    • G01N33/005—H2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90238—Oxidoreductases (1.) acting on hydrogen as donor (1.12)

Definitions

  • the invention relates to enzymatic hydrogen detection systems and related detection methods.
  • H 2 hydrogen
  • H 2 is a colorless, odorless gas, and is also a flammable gas with a lower explosive limit of about 4% in air. Therefore, reliable H 2 sensors are required to detect possible leaks wherever H 2 is produced, stored, or used.
  • sensors that consist of a palladium alloy Schottky diode on a silicon substrate are known.
  • MOS metal-oxide-semiconductor
  • the gas sensing MOS structures are composed of a hydrogen-sensitive metal (palladium or its alloy) deposited on an oxide adherent to a semiconductor.
  • This hydrogen sensor has been commercialized and exploited in detecting H 2 leak during pre-launches of space vehicles.
  • Other research groups have used palladium or the like as a sensing element for detecting H 2 .
  • a hydrogen sensor containing an array of micromachined cantilever beams coated with palladium/nickel has also been reported.
  • Semiconductors e.g. gallium nitride
  • With wide band- gap have also been used to make MOS diodes for H 2 detection.
  • Hydro genases are a class of enzymes that catalyze the interconversion of H 2 and protons (H + ). They can be found in many different microorganisms including Thiocapsa roseopersicina, Allochromatium vinosum, Clostridium pasteurianum, Ralstonia eutropha, and others.
  • the two major classes of hydro genases contain a dimetal active site consisting of an Fe-Fe or a Ni-Fe cofactor.
  • one or more iron-sulfur (Fe-S) clusters play a role in mediating electron transfer from the active site to the protein surface.
  • the active sites of a hydrogenase often contain nickel (Ni) or nickel-iron (Ni-Fe) catalytic centers.
  • Ni-Fe nickel-iron
  • Fe-S iron-sulfur clusters
  • a hydrogen sensor includes a reagent well containing a solution that includes a hydrogenase and an electron acceptor, wherein molecular hydrogen (H 2 ) contacting the solution participates in a redox reaction.
  • H 2 oxidation is catalyzed by the enzyme hydrogenase and the resulting electrons are transferred to the electron acceptor to form a reduced form of the electron acceptor.
  • a detector having a sensing element is disposed proximate to or in the solution. The detector detects the presence of H 2 based on a measurable concentration dependent property of the solution associated with the redox reaction where measurement is taken during or after contacting hydrogen with the solution.
  • the change can be an increase in a concentration of the reduced form of the electron acceptor, which can be correlated with a measurable signal, such as oxidation current or optical intensity.
  • the detector can be an electrochemical detector to measure the oxidation current generated in the redox reaction between H 2 and the electron acceptor, which may be a viologen derivative, methylene blue, cytochromes, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide or flavin adenine mononucleotide.
  • the solution can be aqueous or as a paste or gel material.
  • a redox mediator such as an enzyme cofactor, a ferrocene derivative, an organic dye, ferric cyanide, or a Ru-complex can be included in the solution.
  • the detector can generate a signal based on a color change.
  • the sensing element and the reagent well can be in an integrated chip-based niicrofluidics sensor.
  • the detector can be coupled to a processor with memory to store calibration curve data between the oxidation current and the H 2 concentration and process the measured current to output a H 2 concentration.
  • a method of detecting H 2 involves exposing a gas containing H 2 to a hydrogenase solution with an electron acceptor where a property change of the solution corresponding to the redox reaction is detected and measured.
  • the measurable property can be the oxidation current and resulting from the redox reaction to give a reduced form of the electron acceptor which is then correlated to the H 2 in the gas exposed to the solution.
  • Figures l(a)-(c) show schematic diagrams of several exemplary integrated H 2 sensing systems which can be conveniently mounted in locations of interest, such as near H 2 sources and associated H 2 supply lines.
  • Figure 2 shows a plurality of H 2 .detection systems according to the invention positioned at several locations along a H 2 supply line which provides fuel to an electrochemical generator, such as a PEM fuel cell.
  • an electrochemical generator such as a PEM fuel cell.
  • Figure 3 shows a schematic of a gas flow system used for experimental sample collection and delivery.
  • Figure 4 shows current-time curves obtained in chronoamperometry for 1 and 5% H 2 gas mixtures.
  • Figure 5 shows a Lineweaver-Burke plot of the reciprocal of the reaction rate as a function of the reciprocal of the substrate concentration.
  • Figure 6 shows calibration curves between current and percentage of H 2 in gas mixtures.
  • a hydrogen sensor comprises a reagent well containing a solution that includes a hydrogenase and an electron acceptor, and a detector that can detect a reagent or product of a redox reaction with hydrogen.
  • the reagent well is a partially enclosed orifice constructed with an appropriate shape and of sufficient size to house a portion or all of the detector and contain the solution in a manner that a sufficient contact time between the reagents in solution and the H 2 to achieve a sufficient signal from the detector.
  • the reagent well can be constructed from a material such as a metal, glass, silicon, a plastic or a rubber.
  • the hydrogenase catalyzes the oxidation of hydrogen (H 2 ) gas which contacts or enters the solution contained in the well by the electron acceptor to form a reduced form of the electron
  • a reductant such as ⁇ -nicotinamide adenine dinucleotide
  • NADH (NADH) (which is the reduced cofactor of the soluble hydrogenase from Ralstonia eutropha), is generally also provided.
  • a reductant is generally necessary for the reductive reactivation of the hydrogenase to maintain the desired catalytic effect.
  • the solution is generally an aqueous solution.
  • a detector having a sensor is disposed proximate to or in the solution.
  • the detector permits the determination of a measurable quantity of hydrogen through detection of at least one concentration dependent property of the solution during or following the redox reaction.
  • detection can result from a change in the concentration of a reaction product such as the reduced form of the electron acceptor.
  • Properties of the solution that can be measured include: an oxidation current, a change in the color of the solution, and/or a change in an optical property such as luminescence.
  • Advantages of the enzyme-catalyzed system for the detection of H 2 according to the invention include the ability to operate under ambient conditions and provides high selectivity due to the specificity of the enzyme-based reaction utilized.
  • Hydro genenases are enzymes capable of catalyzing the oxidation of hydrogen. Although the invention is described using the O 2 -tolerant soluble hydrogenase (SH) of Ralstonia eutropha, a wide variety of hydro genenases may be used with the invention.
  • the hydrogenase may be from natural or engineered sources.
  • the electron acceptor can be various materials.
  • the electron acceptor can be selected from viologen derivatives (e.g., benzyl viologen and methyl viologen), methylene blue, cytochromes, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, and flavin adenine mononucleotide.
  • viologen derivatives e.g., benzyl viologen and methyl viologen
  • cytochromes nicotinamide adenine dinucleotide
  • nicotinamide adenine dinucleotide phosphate flavin adenine dinucleotide
  • flavin adenine dinucleotide flavin adenine dinucleotide
  • flavin adenine mononucleotide flavin adenine mononucleotide
  • the soluble hydrogenase (SH) of Ralstonia eutropha which possesses high H 2 -oxidizing activity and O 2 tolerance is used along with the cofactor NADH.
  • SH is a tetrameric Ni-Fe hydrogenase containing several Fe-S and two flavin mononucleotide (FMN) clusters as cofactors in the H 2 oxidation reaction.
  • Gas samples can be collected for more efficient sensing as compared to simple exposure of gaseous H 2 to the solution by several methods, hi one embodiment, a gas sample suspected of containing H 2 is bubbled through the solution that contains the enzyme, a cofactor and an electron acceptor (e.g. BV 2+ ).
  • a gas sample is pumped and mixed with aqueous reagents, such as using T-shape or Y-shape intersections, where the reagent and catalyst contact the gas sample at the intersection.
  • Benzyl viologen (BV) also provides a color change upon reduction.
  • the redox reaction between H 2 and BV 2+ catalyzed by the hydrogenase SH can be represented by equation 1 as shown below.
  • H 2 sensing systems can be assembled from a plurality of separate components, or be miniaturized based a highly integrated embodiment that utilizes a microfabricated device, such as disposed on a chip, for most or all of the system components. Miniaturized total chemical analysis systems have been extensively used for a variety of applications (see Manz, A.; Graber, N.; Widmer, H. M. Sensors and Actuators B 1990, 1, 244-248; Fredrickson, C.
  • microfluidic devices allow for efficient interactions between the gas and aqueous phases, generally leading to much shorter gas exposure time and improved sensitivity.
  • Efficient chemical reactions between gas and liquid samples have been reported in microfluidic systems (see Kobayashi, J.; Yuichiro,'M.; Kuniaki, O.; Akiyama, R.; Ueno, M.; Kitamori, T.; Kobayashi, S. Science 2004, 304, 1305-1307), demonstrating the ability to realize a practical miniaturized sensor system according to the invention based on an enzymatic reaction and electrochemical and/or optical detection.
  • the device can include a well or channel having electrodes therein for holding the reactants, a hydrogenase catalyst and optional cofactor or reductant, a potentiostat, a micro-electro-mechanical system (MEMS) pump, microfluidic pump, or a fan for forcing ambient gas samples into the well, a processor, memory and optional alarm circuitry, all or a part of them interconnected on-chip.
  • Figure l(a)-l(c) shows schematic diagrams of exemplary integrated sensing systems which can be conveniently mounted in locations of interest, such as near H 2 sources and associated H 2 supply lines. Such systems provide continuous, automatic and real time (or near real-time) detection of H 2 in the surrounding environment.
  • system 100 includes at least one reagent well 102 built into substrate die 101. Electrodes 105 and 106 are disposed in well 102. Reagent well 102 contains a solution including a hydrogenase, a cofactor, an electron acceptor and an electrolyte. A potentiostat circuit 107 is electrically coupled to electrodes 105 and 106. An external fan 109 can also be provided for forcing ambient gas samples into the system. The forcing of ambient gas or a reagent solution into the system can also be accomplished using a variety of external pumps, or fans or pumps disposed on the chip. Well 102 has inlet 103 and gas outlet 104.
  • the energy requirements to power various components of the sensing system can be provided by electrical service or by a battery.
  • the battery is preferably a high energy density secondary battery, such as a lithium ion or lithium metal based battery.
  • the power source and the electrical connections are not shown.
  • Potentiostat 107 is disposed on die 101 and is electrically connected to the solution having electrodes 105 and 106 in well 102.
  • the electrodes can be fabricated using screen- printing and other microfabrication techniques, including coating, deposition, sputtering, and photolithographic patterning.
  • the current based electrochemical detection signal associated with the electrochemical reaction sensed by the potentiostat 106 is communicated to a processor 108.
  • the processor 108 can include associated non- volatile memory, such as for storing data manipulation algorithms, predetermined user programmable setpoints, or data taken. Upon detection of more than a predetermined level of H 2 indicating possible danger, system 100 can provide a visual display or an audible alarm (not shown).
  • FIG. l(b) and (c) show alternate exemplary system embodiments based on micro fluidics.
  • the system 110 shown in Fig. l(b) includes a sensing element 115 disposed in solution contained in a reagent well 112 built into a substrate die 111.
  • a gas inlet 113 and outlet 114 are positioned at the ends of the reagent well 112.
  • the sensing element 115 can be an electrochemical detector, a color sensor, or a light detector.
  • Sensing element 115 is electrically connected to an electrical contact 117 disposed on the surface of the die. Again, the electrical contact permits the communication with a processor 118 and a fan 119 or another device can be used to promote the mixing of the gas with the solution.
  • Figure l(c) shows a micro fluidics sensing system 120 according to the invention based on a Y-shape intersection were a gas entering one inlet 122 mixes with a reagent solution entering another inlet 121. Pumps or fans (not shown) can be used for introduction of gas and/or solution.
  • Sensing element 125 connected to electrical contact 127, is disposed in the channel to detect the reaction products of gas mixed with the reagents and catalyst before an outlet 124 for gas and reagent solution. Again the electrical contact 127 is in communication with a processor 128. The reagent solution can be recycled back to the solution inlet port 121 or collected as waste.
  • Figure 2 shows a plurality of H 2 detection systems 100 positioned at several locations along a H 2 supply line which provides fuel to an electrochemical generator 210, such as a PEM fuel cell.
  • Valve 220 when closed turns of the supply of H 2 to the electrochemical generator.
  • the detection of H 2 above a predetermined level can initiate a sequence of events that closes valve 220.
  • NADH nicotinamide adenine dinucleotide
  • BV 2+ benzyl viologen
  • hydrogen 99.99% pure
  • nitrogen industrial grade
  • oxygen industrial grade
  • the enzyme was incubated with NADH for 5 minutes before initiating each H 2 detection trial in order to reactivate the enzyme. This step was implemented last in the solution preparation phase to control the incubation time before H 2 exposure, as well as to prevent premature oxidation of NADH by dissolved oxygen.
  • R. eutropha cells were cultivated heterotrophically at 30 0 C in a mineral medium and stored at -7O 0 C.
  • the soluble hydrogenase (SH) was purified at 4 0 C in air (as described by Burgdorf, T.; van der Linden, E.; Bernhard, M.; Yin, Q. Y.; Back, J. W.; Hartog, A. F.; Muijsers, A. O.; Koster, C. G. d.; Albracht, S. P. J.; Friedrich, B. J. Bacteriol 2005, 187, 3122-3132.
  • the purified SH was dissolved in either 50 mM Tris/HCl (pH 8.0) or 50 mM potassium phosphate (pH 7). The purity of the samples is described below. Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard. The SH samples were stored at -7O 0 C after being received in dry ice or liquid nitrogen. [00034] Three different enzyme batches were used in this study. Batch A SH was prepared using ammonium sulphate precipitation and anionic exchange chromatography. The purified sample was further concentrated by ammonium sulphate precipitation, and then stored in liquid nitrogen. The enzyme concentration of stock solution, batch A, is 113.3 mg/mL in 50 niM potassium phosphate buffer pH 7 while its enzymatic activity was measured 24.4
  • Batch A SH was used in all experiments except where specified.
  • Batch B SH was further purified by hydrophobic interaction chromatography and finally concentrated by ultifiltration (100 kDa cutoff).
  • the enzyme concentration of the stock solution, batch B is 60.0 mg/mL in 20 niM Tris-HCl buffer pH 8 while its enzymatic activity was 31.2 U/mg.
  • Batch C used the same SH as in batch B, except that the enzymatic activity was greater.
  • the enzyme concentration of batch C was 42.6 mg/mL in 20 mM Tris-HCl pH 8.0 and 5% v/v glycerol, while its enzymatic activity was 32.6 U/mg.
  • Detection of H 2 in a gas sample was carried out by exposing the sample to 1 mL of an aqueous solution in a 5 mL flow cell. Full interaction between the gas and solution was ensured by bubbling the gas sample through the solution. Gas bubbling was accomplished by placing a pipette tip on the gas inlet line and submerging it in the enzyme solution to promote H 2 diffusion. A magnetic stirring bar was also used to facilitate thorough mixing of the solution.
  • the flow cell composed of a 5-mL glass beaker, was sealed at the top via a custom-made removable aluminum cap and o-rings. In the cap, five ports allowed for access of gas inlet/outlet and three electrodes for electrochemical detection. Hydrogen was oxidized by hydrogenase and BV 2+ , and the product, BV + , was then detected via chronoamperometry. Gas agitation and stirring were turned off when chronoamperometry was performed. To minimize any possible effects of ambient temperature variation on detection, the flow cell was placed on a hot plate that maintained the temperature at 30°C. It was found that control
  • Chronoamperometry was carried out using a polished, flat disc gold working electrode (diameter of 2 mm), a platinum wire counter electrode, and an Ag/ AgCl (saturated KCl) reference electrode, all of which were obtained from CH Instruments, Inc.
  • the three electrodes were placed equidistant from one another in the solution.
  • the voltage control and current measurement were performed using CH Instruments' 600B electrochemical analyzer and the corresponding software run on a desktop computer.
  • Single-step chronoamperometry was performed at a potential of 0.05 V against the reference electrode.
  • the data acquisition was at a rate of 1.0 kHz.
  • the preparation of gas samples and their delivery to the flow cell are illustrated in Figure 3.
  • a 1 L gas chamber was used to collect and mix gases supplied from N 2 and H 2 cylinders (or O 2 cylinder that is not pictured) at a predetermined ratio. The ratio was determined by the pressure of the gas chamber when the collection of each gas was completed. Therefore, the percentage of H 2 reported is partial pressure, which can be correlated to volume or mass ratio via the idea gas law.
  • the sample chamber was sealed and allowed to sit for a minimum of 10 minutes to ensure complete mixing.
  • AU gas lines and the flow cell were purged with pure N 2 before the experiments.
  • the delivery of the prepared gas sample was carried out using a pressure controller (Model no. 68502-10) from Cole Parmer.
  • Two gas lines controlled by ball valves (Model SS43S4 from Swagelok) and connected to the inlet of the pressure controller, allowed for fast switching between pure N 2 from a cylinder (for system purging) and the gas sample from the gas chamber.
  • the gauge pressure of gas to the flow cell was 1.0 ⁇ 0.1 pounds per square inch (psi) while the flow rate was 20 ⁇ 1 (ml/min).
  • This setup allowed establishment of a constant H 2 partial pressure and facilitated equilibrium of H 2 between gas and liquid phases during the course of a given detection trial.
  • a valve was placed at the inlet of the flow cell, so that the gas flow can be quickly switched off after a certain period of time.
  • the accumulated BV + can be electrochemically detected in the undisturbed solution.
  • a second valve was placed at the gas outlet of the flow cell to control the fiowrate by varying resistance in the gas line.
  • a bubbler was placed in the gas vent line to prevent • back flow and visibly indicate a proper fiowrate. The gas from the bubbler was vented to a fume hood with appropriate ventilation since H 2 leakage into laboratory air must be prevented to avoid possible ignition of hydrogen with ambient air.
  • C/mol A is electrode area
  • D diffusion coefficient
  • C BV + the concentration of BV + .
  • the current measurement was taken at a fixed time as discussed below. Since other parameters are constant, the measured current, i, is proportional to the concentration of BV + in the solution.
  • substrate (S) can be represented by E + S -> ES -» E + P, where P is the product.
  • reaction can be characterized by equation (3) below.
  • V is the rate of disappearance of the substrate (S) or the rate of formation of the product (P)
  • V max is the maximum rate of reaction at a given enzyme concentration
  • K m is the Michaelis constant.
  • the products of the H 2 oxidation include H + and electrons. Since the concentration of H + is essentially constant in a buffer solution, the rate of enzymatic reaction should correspond to the rate of creation of electrons, which are accepted by BV 2+ to produce BV + . When an excess amount OfBV 2+ is present, the rate of the enzymatic reaction is then equal to the rate of formation of BV + .
  • the rate of BV + formation can be calculated from the
  • V ⁇ £ ⁇ L (4)
  • Equation (1) refers to the fact that each mole of H 2 produces 2 moles of protons and electrons, which in turn generate 2 moles of BV + .
  • equation (5) is obtained which describes the relationship between the measured current (i) and the concentration of substrate [S].
  • equation (8) is obtained.
  • the measured CA current at a fixed time is linearly proportional to the partial pressure of H 2 in a gas sample.
  • the enzymatic reaction coupled with chronoamperometry can be used to generate a calibration curve between the current signal and the amount OfH 2 .
  • a measured current signal can be converted to an amount of H 2 , thus providing a sensing mechanism for the presence of H 2 .
  • H 2 gas samples were prepared by pre-mixing H 2 with N 2 and/or O 2 at a pre-determined ratio. Each sample was introduced into the flow cell where electrochemical detection takes place, as illustrated in Figure 3. It is noted that the arrangement shown in Fig.
  • the flow cell included an aqueous solution including hydrogenase, the cofactor NADH for reductive reactivation, and BV 2+ as an electron acceptor.
  • oxidation of H 2 is catalyzed by hydrogenase and the released electrons are accepted by BV 2+ , which is then reduced to BV + .
  • the accumulated amount of BV + after a certain period of time is detected using chronoamperometry and the measured current is used to correlate with the concentration of H 2 in the gas sample.
  • Exposure time was chosen based on the empirically-determined minimum time that produced a clear signal at the desired H 2 concentration range. Gas flow was halted during CA measurements. For each detection trial, a single value for the net signal was determined as the difference in current (averaged over 10 milliseconds) between the background and signal measurements at ⁇ 3 seconds, where the change in current is ⁇ 5% of the final current. The difference in the current between the signal and background is proportional to the difference in the analyte (BV + ) concentration between the H 2 -exposed and blank solution.
  • BV + analyte
  • OCP open circuit potential
  • Figure 4 shows that a significant signal was attained at 1% H 2 gas concentration; and current increases when H 2 concentration changes from 1% to 5%.
  • the background signal was close to 0 A, as expected, with negligible variation in comparison to that of the after-H 2 measurements. Variation in signal increased with H 2 % but, for a given net signal, consistently represented ⁇ 7% of the mean value. This compares well with the estimated error in calculated H 2 liquid concentration of the flow cell of ⁇ 5% (based on error in gas pressure control and the flow rate).
  • a series of substrate concentrations i.e., dissolved H 2 in solution
  • the concentration of dissolved H 2 in solution was estimated using Henry's law (equation 7). Henry's law constant was calculated using the solubility of H 2 in water (2.14 cnrVlOO mL) at 1 atmosphere and the density of H 2 at room temperature (0.0899 g/L).
  • the reaction rate is equal to the rate of formation of BV + , which is determined by the Cottrell equation as discussed above.
  • FIG. 5 shows the Lineweaver-Burke plot obtained from the experimental results.
  • Linear regression resulted in a slope and intercept, from which calculated to be 4.90 x 10 "5 M/s and 3.08 x 10 "3 M for V max and K m , respectively. This result confirmed the assumption when equation (5) was simplified into (6), in which it was noted that the substrate concentration is much lower than K m .
  • the activity of the enzyme was calculated to be 0.9 U/mg at 9% H 2 , which is lower than the value (24 U/mg) reported for SH type A using the optical approach.
  • the discrepancy could be due to two major factors. One is the difference in the methods used.
  • the activity value was calculated from the results from the H 2 -NAD + reaction, which may behave differently as mentioned above concerning the Michaelis-Menten model. Under optimal assay conditions the enzymatic activity using H 2 -BV 2+ assay is about 40% of that using H 2 -NAD + assay.

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Abstract

Selon l'invention, un détecteur d'hydrogène est mis en oeuvre pour détecter et mesurer un produit de réaction en fonction de la consommation d'hydrogène déterminée par une réaction d'oxydoréduction utilisant une hydrogénase et un accepteur d'électrons en solution. La détection est réalisée au moyen d'un capteur disposé à proximité de ou dans la solution. La quantité d'hydrogène présente est mesurée grâce à un signal émis consécutivement à une modification de la concentration de la forme réduite de l'accepteur d'électrons, laquelle mesure est corrélée à la quantité d'hydrogène en contact avec la solution. Cette mesure peut être celle d'un courant d'oxydation résultant de la réaction d'oxydoréduction ou de l'intensité optique d'un produit ou d'un réactif présent dans la réaction d'oxydoréduction. Le détecteur peut être couplé à un processeur qui met en oeuvre une mémoire servant à stocker des données d'étalonnage entre le courant d'oxydation ou l'intensité optique mesurée et la concentration de H2, et traite le signal provenant du détecteur pour produire une concentration d'hydrogène. Le capteur est la cupule de réactif peuvent être intégrés à un capteur de microfluides à puce.
PCT/US2006/009495 2005-03-16 2006-03-16 Detecteur d'hydrogene utilisant une reaction catalysee par des enzymes WO2006101963A1 (fr)

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Cited By (3)

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WO2011027718A1 (fr) * 2009-09-01 2011-03-10 国立大学法人神戸大学 Procédé et trousse pour mesurer les activités enzymatiques de différentes espèces moléculaires du cytochrome p450, d'une manière exhaustive et avec un rendement élevé
DE102016123700A1 (de) * 2016-12-07 2018-06-07 Endress+Hauser Conducta Gmbh+Co. Kg Sensor zur Bestimmung einer von einer Konzentration reaktiver Sauerstoffspezies abhängigen Messgröße
CN115531790A (zh) * 2022-10-08 2022-12-30 南开大学 一种剧毒紫精类化合物的降解方法

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JP2002214190A (ja) * 2001-01-12 2002-07-31 National Institute Of Advanced Industrial & Technology 定電位電解式水素センサ

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