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WO2019229086A1 - Capteur bioenzymatique électrochimique pour mesurer le h2s dans des fluides biologiques - Google Patents

Capteur bioenzymatique électrochimique pour mesurer le h2s dans des fluides biologiques Download PDF

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Publication number
WO2019229086A1
WO2019229086A1 PCT/EP2019/063872 EP2019063872W WO2019229086A1 WO 2019229086 A1 WO2019229086 A1 WO 2019229086A1 EP 2019063872 W EP2019063872 W EP 2019063872W WO 2019229086 A1 WO2019229086 A1 WO 2019229086A1
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Prior art keywords
sqr
enzyme
sulfide
electrode
functional
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Inventor
Frédéric Bouillaud
Véronique BALLAND
Benoit Limoges
Bruno Miroux
Abbas ABOU-HAMDAN
Lindsay ROSSET
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
Universite Paris Diderot Paris 7
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
Universite Paris Diderot Paris 7
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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
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • 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.)
    • G01N2333/90212—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)

Definitions

  • the present invention provides an enzyme-based electrochemical biosensor that can be used for detecting specifically low levels of hydrogen sulfide (H2S) in biological fluids.
  • This biosensor typically includes an electrode system and a functional Sulfide Quinone Reductase (SQR) enzyme.
  • SQR Sulfide Quinone Reductase
  • the present invention also provides methods and systems for measuring H2S in biological fluids by means of this sensor.
  • Hydrogen sulfide is a toxic smelly gas that inhibits the complex IV of the mitochondrial respiratory chain and thus prevents energy production by oxidative phosphorylation.
  • this gas endogenously produced at low levels, acts as a potential neuromodulator and impacts on many important physiological functions like CO and NO.
  • H2S hydrogen sulfide
  • WO 2014/138999 proposes to detect H2S producing bacteria in the gut of patients suffering from an Inflammatory Bowel Disease, in order to indirectly assess the level of H2S in the gut.
  • WO 2015/109221 proposes to use recombinant micro-organisms overexpressing sulfide metabolizing enzymes to treat tainted gas feedstock that has been contaminated by a S substrate such as H2S.
  • H2S The most widely used method to determine H2S is the methylene blue method (Stipanuk and Beck, 1982). It has the advantage of being easy to set up and use as it consists of a rather simple colorimetric procedure that requires determination of optical density in a spectrophotometer at the end of the procedure.
  • Major drawbacks include a high limit of detection (>1 mM), interference with colored substances and a non-linear behavior of the calibration curve.
  • the method measures not only H2S, but also sulfide from iron-sulfur centers that are ubiquitous in biological systems, because of the acidity of the reactants used in the procedure (Papapetropoulos 2015).
  • This method therefore determines not only the amount of free sulfide present in a biological fluid, but also the amount of "releasable sulfide" present in this sample. This explains why high levels micromolar ranges of H2S are measured using this method, even though such a concentration would be toxic for the biological systems and said forms could not be stable in presence of metabolically active cells (Lade et al, 2010; Abou Hamdan et al, 2015).
  • Electrode-based systems have been proposed to try to solve this problem.
  • EhS-sensors namely ISO-H2S-2 and ISO- H2S-100. They contain an electrolyte (preferably a sodium carbonate buffer solution of pH 10 containing propylene carbonate and potassium ferricyanide) and a membrane of silicon polycarbonate having a thickness of 10 to 50 micrometers (aimed to be H2S permeable).
  • Another electrode system called the “Sulfidostat”, has been built by Searcy et al. (2004) to measure sulfide consumption (i.e. a relative value).
  • the electrode used in this system is based on the reactivity of a silver electrode toward sulfide; nevertheless it is likely that other ions reactive with silver would hampered selectivity of measures.
  • H2S levels can be evaluated by measuring H2S metabolites (thiosulphate and sulphate) as surrogate markers.
  • H2S metabolites thiosulphate and sulphate
  • This approach to measuring H2S has not been used to any significant extent as it awaits validation that H2S metabolites can indeed accurately reflect H2S levels.
  • enzymatic activity assays for cystathionine-g lyase and cystathionine-b synthase have been reported, these are rarely used (Papapetropoulos 2015).
  • BOX 1 of Wallace JL et Wang R, 2015 pages 332
  • the reliability and accuracy of all these H2S measurement systems are strongly contested by the technical experts in the art.
  • the sensors and probes developed to date are unable to measure endogenously produced H2S present in biological samples (e.g., in plasma, tissues, homogenates or cell culture supernatants). Only relative differences in H2S levels can be, in particular conditions, reliably detected.
  • the present invention is based on the finding that it is possible to use a recombinant Sulfide Quinone Reductase (SQR) enzyme that is active and functional to generate a detectable electric signal that is proportional to the amount of H2S present in a solution, even for very low level of H2S.
  • SQL Sulfide Quinone Reductase
  • SQR Sulfide Quinone Reductase
  • Recombinant SQR can be used in electrochemistry experiments where it generates an electrical signal proportional to the amount of sulfide that is present in an aqueous solution.
  • the originality of the present invention consists on the one hand in using an enzyme having high specificity and sensitivity toward hydrogen sulfide (enzymatic reactions are far better than any physicochemical methods) and, on the other hand, in coupling this enzyme to a physical sensor system.
  • SQR has several advantages since this enzyme is dedicated to control the concentration of hydrogen sulfide in the body.
  • SQR has a high sensitivity toward H2S (the inventors have shown that the mammalian SQR reacts at H2S concentrations of the order of 10 to 100 nM, see Lastill et al, 2010).
  • SQR has a better specificity for H2S than any other methods and this specificity is working even in complex biological environment.
  • the present invention relates to the use of a functional Sulfide Quinone Reductase (hereafter also called "SQR”) enzyme for detecting hydrogen sulfide (H2S) electrochemically in a biological fluid.
  • SQL Sulfide Quinone Reductase
  • sulfide include here three forms whose proportions vary according to the pH: the H2S gas and its ionized forms bisulfide (HS ) and sulfide (S 2 ).
  • biological fluid refers to any samples which have been obtained from a patient and which is fluid or viscous. It can be for example a biological fluid such as urine, cerebrospinal fluid, pleural fluid, synovial fluid, peritoneal fluid, amniotic fluid, gastric fluid, blood, serum, plasma, lymph fluid, interstitial fluid, saliva, physiological secretions, tears, mucus, sweat, milk, semen, seminal fluid, vaginal secretions, cell lysates, fluid from ulcers and other surface eruptions, blisters and abscesses.
  • the said biological sample can be tested raw or pre-treated prior to use. Pre-treatment herein means e.g., preparing plasma from blood, diluting viscous fluids, filtering or the like.
  • said biological fluid is chosen from whole blood, serum and plasma.
  • detecting encompasses the process of revealing the presence (or the absence) of the target metabolite in the sample and also the process of "analyzing the amount”, “measuring the concentration”, “measuring the variations of concentration”, “measuring the appearance or the disappearance” and “quantifying” the said metabolite.
  • the detection of hydrogen sulfide is performed "electrochemically" for example by voltammetry or by amperometry.
  • the detection can be performed when the SQR enzymes are in solution, or when they are confined to the surfaces of electrodes.
  • the present application proposes the use of a biosensor including an electrode system and a solution containing a functional Sulfide Quinone Reductase (SQR) enzyme.
  • a biosensor including an electrode system and a solution containing a functional Sulfide Quinone Reductase (SQR) enzyme.
  • SQL Sulfide Quinone Reductase
  • the present application proposes the use of a biosensor including an electrode system onto which a functional Sulfide Quinone Reductase (SQR) enzyme has been concentrated.
  • SQL Sulfide Quinone Reductase
  • SQR enzymes (EC 1.8.5.4) are found widely among bacteria and in some groups of eukaryotes. They are part of the flavoproteins family with a molecular mass of about 47-50kDa that can be monomers or can assemble into dimers or trimers in solution. They contain one Flavin adenine dinucleotide (FAD) cofactor per monomer and are associated with the prokaryotic cytoplasmic membrane or the mitochondrial inner membrane. They catalyze the following chemical reaction: n HS _ + n quinone -> polysulfide + n quinol
  • FAD Flavin adenine dinucleotide
  • SQR couples the oxidation of sulfide to the reduction of quinone via the intermediacy of an active site persulfide.
  • the acceptor quinone cosubstrate can be for example ubiquinone, plastoquinone or menaquinone.
  • SQR enzymes from various species have been characterized and their activity in the sulfide oxidation pathway well studied (in human, see Jackson M R et al., 2012 and Mishanina TV. et al, 2015; in worm, see Theisen U & Martin W., 2008; in bacteria, see Marcia M. et al, 2010).
  • the SQR enzymes used in the present invention do not catalyze the transformation of succinate into fumarate. They have no succinate deshydrogenase enzymatic activity (EC 1.3.5.1).
  • Any enzyme having a Sulfide Quinone Reductase activity can be used in the present invention.
  • these polypeptides will be referred to as "functional SQR enzymes”. Said polypeptides therefore encompass any active fragment, chimeric protein or recombinant protein exhibiting said activity.
  • the functional SQR enzyme used in the invention is the human SQR of SEQ ID NO:7 (reference UniprotKB: Q9Y6N5 (SQOR_HUMAN)
  • the functional SQR enzyme used in the invention is a bacterial SQR, for example the SQR enzyme of Aquifex aeolicus of SEQ ID NO:5, encoded by SEQ ID NO:6 (reference UniprotKB:067931, code PDB: 3HYV).
  • thermostable SQR of the bacterium Aquifex aeolicus that was finally chosen and used in the examples.
  • This thermostable SQR enzyme has indeed been characterized biochemically and spectroscopically and its tridimensional structure has been resolved (Marcia et al, 2009).
  • Sulfide oxidation takes place on the re side of FAD and the two electrons resulting from this oxidation are therefore donated one by one to the membrane quinone pool located at the si side of FAD.
  • A. aeolicus SQR a water labile and insoluble cyclooctasulfur (S8) was suggested to be released into the membranes through a hydrophobic cavity and to be compartmentalized in the cytoplasm, where A. aeolicus is known to store sulfur globules (Guiral M et al, 2005).
  • variants mutated enzymes
  • fragments of these enzymes provided that they are functional.
  • Chimeric SQRs i.e. functional enzymes reconstituted from several fragments from SQR of different organisms
  • Such variants, fragments, or chimeric SQRs should contain at least the polypeptide sequences that are responsible for the enzymatic activity of SQR. They have preferably a minimal size of 400 amino acids.
  • these variants / fragments / chimeric SQRs would be designed to be less hydrophobic than the wild-type enzyme of the same species. This can be achieved for example by removing or altering the hydrophobic region of the enzymes known to interact with the membrane (Marcia et al., 2009).
  • This hydrophobic region is HVVVIGGGVGGIATAY (SEQ ID NO:8) in the N-terminal sequence of the A. aeolicus SQR.
  • the C-terminal end (residues 376-412) of the A. aeolicus SQR has been shown to bind detergent in the crystal structure and is therefore predicted to interact with the membrane together with N-terminal hydrophobic region (Marcia et al., 2009)
  • any protein moieties (whole proteins, protein fragments or tags), for purification or visualization purposes, provided that these moieties do not affect the functionality of the SQR enzyme.
  • SQR that are tagged with conventional tags (histidine, streptavidine, avidine, neutravidine, FLAG- tag, epitope-tag, SBP-tag, etc.).
  • tags will be covalently coupled to the N-terminal side of the SQR enzyme.
  • Fluorescent proteins such as GFP or chemical dyes can also be coupled to the SQR enzyme.
  • Functional SQR enzymes are able to reduce the quinone coenzyme in the presence of sulfide and/or sulfite.
  • SQR activity can be assessed by any conventional means well known in the art. For example, it is possible to measure this activity by following the reduction of CoQi at the appropriate wavelength, by spectrometry, as described in Mishanina TV. et al, 2015, under the section "SQR activity assays", or by any other means as described in Marcia et al, 2010, or in NObel et al, 2000.
  • Functional SQR enzymes are typically able to reduce CoQi with a k cat value comprised between 1 s 1 and 100 s 1 .
  • the functional SQR of the invention or the variant thereof is produced recombinantly in host cells.
  • Any host cell can be used for this purpose.
  • bacterial host cells are used to produce the functional SQR of the invention or the variant thereof.
  • SQR enzymes, variants and fragments or chimaera thereof should contain the cofactor Flavin Adenine Dinucleotide (FAD) in a one to one ratio with the protein. Therefore, when the SQR enzymes used in the invention are recombinantly produced, it is important to ensure that FAD is present in the producing cells.
  • said producing cells are bacteria and FAD is thus naturally incorporated in the recombinant SQR enzymes.
  • at least 25% of the SQR enzymes contain the FAD factor, more preferably at least 50%.
  • FAD can be added extemporaneously by incubating the enzyme with an excess amount of exogenous FAD. This will compensate the lacked FAD and allow to obtain a solution containing up to 80% and possibly up to 100% functional FAD-containing SQR.
  • the inventors have verified that the presence of unbonded FAD remaining in the enzyme solution (if any) does not affect the activity of the SQR enzyme with bound FAD. This optional complementary step is useful to optimize the amount of active enzyme especially for immobilization on the electrode.
  • the SQR enzymes Due to their hydrophobic and amphiphilic nature, the SQR enzymes are poorly soluble and therefore difficult to purify and manipulate in biological systems. Consequently, the studies in solution require the use of a detergent to disperse the proteins in the medium.
  • Said solubilizing agent is for example a non-ionic detergent that can be for example chosen in the group consisting of: glucosides, maltosides, CYMAL, NPG, sulfoxides, POE-glycols, digitonin, Decanoyl-N-hydroxyethylglucamide and TritonTM X-100.
  • Glucosides are for example: n-Dodecyl-b- D-glucopyranoside (C12G), n-Heptyl ⁇ -D-glucopyranoside (C7G), n-Heptyl ⁇ -D-thioglucopyranoside (HTG), n-Hexyl ⁇ -D-glucopyranoside (C6G), n-Nonyl ⁇ -D-glucopyranoside (NG), n-Octyl-b-O- glucopyranoside (OG) or mixture of alkyl-glucopyranosides (Elugent).
  • Maltosides are for example n-Decyl ⁇ -D-maltopyranoside (DM), n-Dodecyl ⁇ -D-maltopyranoside (DDM), n-Nonyl-b-O- maltopyranoside (NM), n-Nonyl ⁇ -D-thiomaltopyranoside (NTM), n-Octyl ⁇ -D-maltopyranoside (OM), n-Tetradecyl ⁇ -D-maltopyranoside (TDM), n-Tridecyl ⁇ -D-maltopyranoside (Tri-DM), n- Undecyl ⁇ -D-maltopyranoside (UDM), N-Undecyl ⁇ -D-thiomaltopyranoside (UDTM).
  • DM Decyl ⁇ -D-maltopyranoside
  • DDM Dodecyl ⁇ -D-maltopyranoside
  • NM n-Nonyl-
  • CYMAL detergent are for example 4-Cyclohexyl-l-Butyl ⁇ -D-Maltoside (Cymal4), 5-Cyclohexyl-l-Pentyl ⁇ - D-Maltoside (Cymal5), 6-Cyclohexyl-l-Hexyl ⁇ -D-Maltoside (Cymal6), 7-Cyclohexyl-l-Heptyl ⁇ -D- Maltoside (Cymal7).
  • NPG detergent are for example Decyl maltose neopentyl glycol (DMNG), Lauryl maltose neopentyl glycol (LMNG), Octyl glucose neopentyl glycol (OGNG).
  • Sulfoxides are for example 2-hydroxyethyloctylsulfoxide (HESO) or n-Octyl-2-hydroxyethylsulfoxide. It is also possible to use Decanoyl-N-hydroxyethylglucamide (HEGA10) or Igepal CA-630 (Nonidet P-40 substitute).
  • HESO 2-hydroxyethyloctylsulfoxide
  • HEGA10 Decanoyl-N-hydroxyethylglucamide
  • Igepal CA-630 Nonidet P-40 substitute
  • OPOE Tetraethylene glycol monooctyl ether
  • C8E4 or OTOE Tetraethylene glycol monooctyl ether
  • Pentaethylene glycol monodecyl ether C10E5
  • Hexaethylene glycol monodecyl ether C10
  • Said solubilizing agent can also be a zwitterionic detergent chosen from: 3-[(3-Cholamidopropyl)- Dimethylammonio]-l-PropaneSulfonate] (CHAPS), 3-[(3-Cholamidopropyl)-Dimethylammonio]-2- Hydroxy-l-PropaneSulfonate (CHAPSO), n-Dodecyl-N,N-dimethylglycine (Empigen), Fos-choline 10 or Decylphosphocholine (FClO/DePC), Fos-choline 12 or Dodecylphosphocholine (FC12/DPC) Fos- choline 14 or Tetradecylphosphocholine (FC14), N,N-dimethyldecylamine N-oxide (decyldimethylamine oxide) (DDAO), N,N-dimethyld
  • Said solubilizing agent is for example an amphiphatic polymer or "amphipol". These molecules are surfactants that carry a large number of hydrophobic chains. They are able to keep water-soluble hydrophobic proteins such as SQR in their native state, under the form of small hydrophilic complexes (for reviews, see refs. Zoonens and Popot, 2014).
  • the amphipol can be for example chosen in the group consisting of: A835 - Amphipol A8-35, or P5008 - PMAL-C8 (commercialized by Anatrace).
  • the functional SQR enzyme used in the present invention is solubilized in a medium containing the detergent n-Dodecyl- -D-Maltopyranoside (also known as "DDM").
  • the functional SQR enzyme of the invention is suspended in a solution or a buffer (e.g., a Tris-HCI buffer) containing 0.001% to 5% of DDM.
  • the solution or buffer contains between 0.03% and 2% of DDM.
  • soluble redox molecules have to be present for the enzyme to be active, they serve as acceptor of the electrons during the oxidation of hydrogen sulfide.
  • they mediate the electron transfer between the electrode and the FAD cofactor. They are usually called "cosubstrate" of the enzyme.
  • the SQR enzyme can theoretically use several types of cosubstrate, including one or two-electron mediators.
  • the various one-electron acceptors are less preferred because the inventors have found that they react less efficiently with the SQR and also they are able to react directly with the sodium sulfide in the absence of SQR (data not shown).
  • the cosubstrate used in the present invention is a two- electron acceptor.
  • the cosubstrate used in the present invention has a formal potential comprised between -0.2 V and + 0.3 V versus a Normal Hydrogen Electrode (NHE), at pH of 7 and at 30°C.
  • NHE Normal Hydrogen Electrode
  • Said cosubstrate should also be both hydrophobic enough to reach the FAD cofactor of the SQR enzyme and hydrophilic enough to mediate the transport of the electrons, especially when the SQR is used in solution.
  • Bacteria use a mixture of quinones (ubiquinones, napththoquinones, and the derivatives menaquinone and rhodoquinone) for bioenergetics reactions. Therefore the SQR enzyme can use in principle any quinone derivative with a redox potential between -139 and +110 mV versus NHE (Zhang et al. 2016).
  • the cosubstrate used in the invention is a ubiquinone Q n with n comprised between 1 and 10, or a natural derivative thereof, or a synthetic derivative thereof.
  • said cosubstrate is CoQi (2,3-Dimethoxy-5-methyl-6-(3- methyl-2-butenyl)-l,4-benzoquinone). It is also possible to use CoQo (2,3-dimethoxy-5-methyl-l,4- benzoquinone).
  • ubiquinone derivatives When the cosubstrate is used in solution, ubiquinone derivatives must be soluble or solubilized with the detergent mentioned above for SQR purification and in a range of concentration compatible with its activity. Any ubiquinone derivatives can be used when the SQR enzyme and the cosubstrate are immobilized in a polymer on a working electrode (see below).
  • the cosubstrate used in the present invention is the quinone CoQi.
  • biomolecules other than SQR provided that: 1) they have an affinity for sulfide sufficient to recognize it at concentrations below the micromolar.
  • the SQR is an example, yet there are others for example certain respiratory pigments capable of fixing H 2 S with a high affinity.
  • any enzyme that is simply able to oxidize sulfide by reducing FAD such as the enzyme NAD(P)/FAD-dependent oxidoreductase of the Comamonas granuli bacteria provided that the F S can access to the FAD moieties.
  • This enzymatic activity can be assessed by measuring the optical properties of FAD (absorbance and/or fluorescence), as proposed e.g., in Mishanina et al, 2015.
  • the present invention relates to an in vitro method for measuring the concentration of FhS in a biological fluid, said method comprising at least the steps of: i) bringing said biological fluid into contact with a functional Sulfide Quinone Reductase (SQR) enzyme, and ii) electrochemically detecting the activity of said SQR enzyme.
  • SQL Sulfide Quinone Reductase
  • This method optionally comprises the step of determining the concentration of FhS in said biological fluid, based on the detected activity of said SQR enzyme.
  • Embodiment 1 The SQR enzymes are in solution
  • the present invention describes for the first time an electrochemical biosensor system comprising an electrode system and a functional Sulfide Quinone Reductase (SQR) enzyme.
  • this biosensor system can be used to detect and quantify the H2S present in a biological fluid, in a specific, rapid and reliable manner.
  • the present invention relates to a method comprising the following steps: a) put in contact the functional SQR enzyme described above with said biological fluid, in presence of the solubilizing agent described above and in presence of the cosubstrate described above, b) put said solution in contact with an electrode system, c) apply an appropriate voltage between the working and reference electrodes of said electrode system, and d) measure the electric current flowing through said working electrode.
  • the electrode system When the electrode system is placed in the sample containing the biological fluid and the other components of the redox reaction (the functional SQR and its cosubstrate), hydrogen sulfide will be oxidized, the cosubstrate will be reduced and a current will accordingly be generated.
  • the redox current generated by this reaction is proportional to the hydrogen sulfide concentration.
  • the change in the current signal will provide a quantitative indication of the dissolved amount of hydrogen sulfide in the sample.
  • Chronoamperometry which consists of the measurement of current versus time at a constant potential after application of a perturbation, is preferably used when the SQR enzymes are used in a solution. Cyclic or linear voltammetry can be used as well.
  • the electrode system used in said embodiment of the invention contains at least a working electrode and a reference electrode.
  • the electrode system is a typical three electrodes system containing a Ag/AgCI electrode (SCE) as a reference electrode, a carbon-based screen-printed electrode as a counter electrode, and a carbon-based screen-printed electrode (SPE) as a working electrode (1.5 mm diameter), the two latter being prepared from a commercialized carbon ink.
  • SCE Ag/AgCI electrode
  • SPE carbon-based screen-printed electrode
  • Any other reference electrode (compartmentalized behind a porous frit or plug) can be used.
  • the counter and working electrodes can also be made of any other inert materials such as any carbon-based materials or any noble metals.
  • the working and reference electrodes are the same as in three-electrodes system.
  • Any type of working electrode/reference electrode can be used.
  • carbon-based electrodes are used.
  • the method of the invention can be either performed under anaerobic conditions or in O2 controlled atmosphere. It can also be performed in presence of O2, in normal atmosphere.
  • the method of the invention is preferably performed at a temperature comprised between 10°C and 50°C, preferably at about 40°C.
  • This range of temperature can be adjusted to the thermodynamic properties of the SQR enzymes used in the sensor. For example, if the SQR enzyme of the Aquifex aeolicus bacteria is used, then the detection of H S with the sensor of the invention is preferably performed at 40°C.
  • the quantity of the cosubstrate to be added in the solution to be tested is comprised between 10 mM and 200 pM. In a more preferred embodiment, it is comprised between 20 and 80 pM, preferably 40 pM.
  • the voltage applied to the electrode system is between -0.2 V and 0.5 V versus NHE.
  • This voltage is applied by an instrument capable not only of maintaining a constant potential between the working electrode and the reference electrode, but also to measure the current generated between the working and counter electrodes (or the reference electrode if the electrode system is based on a two electrodes system).
  • the instrument can be for example a potentiostat.
  • the calibration of the electrode system is preferably done prior to the testing of the biological sample with standard solutions containing known amount of sulfide (for example comprised between 0 and 100 pM of h S).
  • This calibration step is a classical step for the skilled person.
  • the present invention also relates to a biosensor kit comprising the following features: a) a functional SQR enzyme or a variant thereof (as described above), b) a solubilizing agent (as described above), c) a cosubstrate (as described above), d) an electrode system containing two or three electrodes (as described above), e) optionally, means to apply an appropriate voltage between the working and reference electrodes of said electrode system, and f) optionally, means to measure the electric current flowing through said working electrode.
  • This biosensor kit may also contain standard solutions of sulfide for calibrating the device prior to its use (e.g., Na S solutions), as well as a negative control to check the absence of contaminating sulfide (e.g., ultrapure water).
  • sulfide for calibrating the device prior to its use
  • a negative control to check the absence of contaminating sulfide e.g., ultrapure water.
  • Embodiment 2 the SQR enzymes are immobilized in the vicinity of the working electrode
  • the SQR enzymes and optionally their cosubstrates are physically associated with the working electrode of the above-mentioned electrode system, in order to facilitate the redox reaction and the detection of the generated current. This will make the biosensor operational in all practical situations and give it its full sensitivity.
  • the detection is preferably performed by voltammetry (linear or cyclic) or by amperometry. Chronoamperometry is herein preferred.
  • Conducting polymers contain for example polyacetylene, polythiophene, polyindole, polypyrrole, polyaniline, etc. (see Gerard et al, 2002).
  • the present invention relates to an electrochemical biosensor system comprising an electrode coated or in contact with a reagent layer containing a functional Sulfide Quinone Reductase (SQR) enzyme.
  • SQL Sulfide Quinone Reductase
  • Said reagent layer can be a polymeric layer, a membrane, a self-assembled monolayer (SAM), a resin, or any means that can physically associate the SQR enzyme and its cosubstrate to an electrode.
  • Said means are described above and in Thevenot DR et al, 1999; Gerard et al, 2002; Melin F. and Hellwig P., 2013 and Kulkarni et al, 2016.
  • the reagent layer contains not only the SQR enzymes but also their substrates. Consequently, the enzymes and the cosubstrates are in contact or close to each other, what favors a rapid and efficient redox reaction.
  • the present invention also relates to a method for measuring the concentration of F S in a biological fluid, using said biosensor.
  • Preferred methods consist in: a) putting in contact a working electrode coated with a reagent layer containing the SQR enzyme and the cosubstrate, as described above, with said biological fluid, b) applying an appropriate voltage between the working and reference electrodes of said biosensor, and c) measuring the electric current flowing through the said working electrode.
  • the method of the invention can be either performed under anaerobic conditions or in O2 controlled atmosphere. It can also be performed in presence of O2, in normal atmosphere.
  • the method of the invention is preferably performed at a temperature comprised between 10°C and 50°C, preferably at about 40°C.
  • This range of temperature can be adjusted to the thermodynamic properties of the SQR enzymes used in the sensor. For example, if the SQR enzyme of the Aquifex aeolicus bacteria is used, then the detection of H2S with the sensor of the invention is preferably performed at 40°C.
  • the quantity of the cosubstrate to be added in said reagent layer is comprised between 10 mM and 200 pM.
  • the voltage applied to the electrode system is between -0.2 V and 0.5 V versus NHE.
  • This voltage is applied by an instrument capable not only of maintaining a constant potential between the working electrode and the reference electrode, but also to measure the current generated between the working electrode and the counter electrode (or the reference electrode if the electrode system is based on a two electrodes system).
  • the reagent layer consists in the SQR enzymes themselves, that are physically maintained in close vicinity of the electrodes.
  • the SQR enzymes can be bound to the electrode surface by means of functional groups or spacers.
  • Such functional groups or spacers are well-known in the art. Some of them have been described above and designated as "tags". They rely on the interaction of a group or tag (e.g. biotin) with its interacting partner, which can be present on the electrode (e.g., avidin). Other tags can be used alternatively (SBP-tags, FLAG-tags, His-tags, epitope-tags such as Myc-tag, FIA-tag, Spot-tag, NE-tag) provided that their interacting group is grafted within the vicinity of the working electrode present in the device.
  • the SQR enzymes used in this system are N-terminally fused with said functional group.
  • the cosubstrates of the SQR enzymes are added to the sample to be tested extemporaneously, in a sufficient amount for the current to be detected. These cosubstrates are as described above.
  • the reagent layer contains solid supports onto which the SQR enzymes are bound, said solid supports being preferably maintained in close vicinity of the electrodes by physical or magnetic means.
  • the SQR is bound to magnetic beads that are maintained closed to the electrodes with the help of a magnet.
  • the magnetic enzyme coated beads are collected as a film on the surface of the electrode and the catalytic currents can be recorded by voltammetry or amperometry. This system enables to concentrate the SQR enzymes close to the electrode and therefore enhance the catalytic current responses.
  • the cosubstrate of the SQR enzymes has to be added to the sample to be tested extemporaneously, in a sufficient amount for the current to be detected (e.g. 40 mM of CoQi).
  • a sufficient amount for the current to be detected e.g. 40 mM of CoQi.
  • Other cosubstrates can be used, as described above.
  • the reagent layer mentioned above contains magnetic beads onto which the SQR is bound by means of functional groups or spacers.
  • the functional groups or spacers disclosed above for the binding of the enzymes directly to the electrodes can be used in this respect.
  • the SQR enzymes used in this embodiment are N-terminally fused with said functional group and bound to magnetic beads that have been coated with the interacting partner of said functional group.
  • the SQR enzymes have been tagged with the Strep-tag ® , more preferably at their N- terminal side, and the magnetic beads have been coated with streptavidin.
  • SQR enzymes that have been tagged with Streptavidin, more preferably at their N-terminal side, and magnetic beads that have been coated with biotin or the Strep-tag ® .
  • Any type of magnetic beads or particles can be used in this specific embodiment. Said beads or particles have preferentially a diameter comprised between 10 nm and 5 pm, more preferentially a diameter comprised between 50 nm and 3 pm, and even more preferentially a diameter comprised between 100 nm and 1 pm.
  • these beads are preferentially functionalized with a group that is able to bind the recombinant SQR enzymes (e.g., biotin or neutravidin).
  • these magnetic beads are streptavidin-coated beads as exemplified below.
  • three-electrodes or two-electrodes systems can be used (see above). Any type of working electrode/reference electrode and counter electrode can be used. Preferably, carbon- based electrodes are used.
  • the working and the counter electrodes can be screen- printed with a carbon-based ink and the reference electrode can be a silver/silver chloride reference electrode in an extension with a porous plug.
  • the potential of the silver couple (Ag + /Ag) depends on HS , it can be necessary to use a true reference electrode wherein the silver couple is isolated from the solution containing F S with a saline bridge (such systems are commercialized for example by WPI, Driref-2). In so doing, the intensity of the current can be measured at fixed potential whatever the concentration of F S is.
  • the working electrode used in this embodiment of the system of the invention is an inert conductive electrode, preferably a carbon-based electrode.
  • the present invention also relates to a biosensor kit comprising solid supports (e.g., magnetic beads) physically associated with SQR enzymes.
  • Said kit may also contain a vial containing the cosubstrate of said SQR enzymes and/or an electrode system.
  • This biosensor kit may also contain positive and negative control standard solutions for calibrating the device prior to its use.
  • Positive control standard solutions are for example 1 M Na2S solutions that are diluted so as to reach sulfide concentrations of 50 nM to 50 mM.
  • Negative control solutions are for example composed of ultrapure water.
  • the cosubstrate of the SQR enzymes will have to be added to the electrode system extemporaneously, for a redox current to be generated.
  • the present invention relates to methods for measuring the concentration of F S in a biological fluid, using the biosensors of the invention and/or the kits of the invention.
  • the preferred methods consist in: a) putting in contact an electrochemical biosensor system in which the SQR enzymes are immobilized onto or close to the electrodes (directly or by means of intermediary solid supports), as described above, with said biological fluid, b) adding a sufficient amount of the cosubstrate of said SQR enzymes in the sample containing the electrodes, c) applying an appropriate voltage between the working and reference electrodes of said biosensor, and d) measuring the electric current flowing through the said working electrode.
  • the method of the invention can be either performed under anaerobic conditions or in O2 controlled atmosphere. It can also be performed in presence of O2, in normal atmosphere.
  • these methods are performed at a temperature comprised between 10°C and 50°C, preferably at room temperature.
  • This range of temperature can be adjusted to the thermodynamic properties of the SQR enzymes used in the sensor.
  • the detection of H2S with the sensor of the invention is preferably performed at 40°C.
  • the quantity of the cosubstrate to be added in the sample containing the electrode is comprised between 10 mM and 200 pM. In a more preferred embodiment, it is comprised between 20 and 80 pM, preferably it is of 40pM.
  • the voltage applied to the electrode system is between -0.2 V and 0.5 V versus ENH
  • This voltage is applied by an instrument capable of maintaining a fixed DC potential between the working electrode and the reference electrode, for example a potentiostat.
  • biosensors of the invention will have immediate applications in research. Their introduction in clinical research should stimulate the use of therapies based on the properties of the physiological H2S mediator. Endogenous H2S would become a measurable and relevant parameter for monitoring the cardiovascular or inflammatory status of patients.
  • H2S donors i.e. hydrogen sulfide releasing agents
  • these biosensors will therefore improve the monitoring of the use of these H2S donors.
  • biosensors of the invention should allow a precise follow-up of the patient and thus allow a local use of the toxic effects or general effects of sedation/anesthesia with H2S.
  • the present invention also relates to the in vitro use of the biosensors and kits of the invention, for monitoring the treatments of cardiovascular and/or inflammatory diseases in patients in need thereof.
  • biosensors and kits of the invention for diagnosing diseases the etiology of which implied H2S levels imbalance such as arthritis, inflammatory bowel disease, multiple sclerosis, myocardial dysfunction and chemoprevention of cancer, in patients in need thereof.
  • the biosensors of the invention can also be used for in vivo monitoring the sulfide levels in patients in need thereof.
  • Present knowledge indicates a wide range for sulfide relevance in physiologic processes. This includes cardiovascular medicine, management of inflammatory state, state of consciousness and memory linked processes.
  • the biosensors of the invention can also be used for measuring the sulfide content present in the environment (water, sea, etc.) or goods.
  • Purified Strep-SQR (3 mM) was incubated with magnetic beads (1 mg/mL) coated with streptavidine to immobilize 400pMol SQR/mg of beads. Assay was performed using 70 mI of SQR-beads in solution. SQR activity was determined by recording the decylubiquinone spectrum after correction at 320nm. black curve: dUQ; dashed grey curve: dUQ+control beads+Na2S; dotted grey curve: dUQ+SQR-beads+Na2S A.
  • Cyclic voltammetry measurements were performed with an AUTOLAB potentiostat in an open electrochemical cell of three electrodes: a saturated calomel electrode (SCE) as a reference electrode, a platinum wire as a counter electrode and a screen printed electrodes 'SPE' (4mm diameter). Measurements were carried out at 40°C in the presence of Coenzyme Q1 (CoQl) as a cosubstrate. Reaction was initiated by the injection of 3 mM of His-tagged (A) or Strep-tagged (B) purified SQR (162 pg) into the reaction mixture containing excess of Na S.
  • SCE saturated calomel electrode
  • B Strep-tagged
  • Cyclic voltammetry was performed at 20 mV.s 1 from -0,5 to +0,4V vs SCE.
  • Black line reaction mix comprising CoQ, SQR, and H S; dashed grey line: without SQR; dotted line, without H S and SQR
  • thermostability of the enzyme allows experiments to be conducted at large range of temperature without any enzyme denaturation. Its tridimensional structure was resolved (Marcia et al, 2009) and it has been characterized either biochemically and spectroscopically (Marcia et al, 2010).
  • Native SQR from Aquifex aeolicus has been well studied biochemically and spectroscopically by measuring the enzyme activity as sulfide-dependent decylubiquinone (dUQ) reduction in a UV/Vis spectrophotometer.
  • dUQ sulfide-dependent decylubiquinone
  • Electron acceptor such as Coenzyme Q1 'CoQl' and decylubiquinone 'dUQ', Sodium Sulfide 'Na2S', bacterial growth medium 2xYT, Isopropyl b-D-thiogalactopyranoside 'IPTG', Glucose oxidase from Aspergillus niger type VII, the cofactor Flavin adenine dinucleotide disodium salt hydrate and catalase from bovine liver was all purchased from Sigma-Aldrich. The detergent n-Dodecyl-b-O- Maltopyranoside 'DDM' was obtained from Anatrace. Restrictions enzymes BamHI, EcoRI and Xhol was purchased from Promega.
  • E. coli DFI5a strain purchased from Invitrogen Life Technology while the heterologous expression of SQR from Aquifex aeolicus was performed using the C45(DE3) strain that comprises premature termination codon within the T7RNAP gene described in Angius et al (2016) but other bacterial strains known from the one with skill in the art are suitable.
  • a synthetic version of the gene encoding sulfide quinone reductase 'SQR' from Aquifex aeolicus was used.
  • the sequence of this synthetic gene is provided in SEQ ID NO:6.
  • This synthetic gene was flanked between BamHI and Xhol restriction sites of the plasmid pEX-K4 provided by Eurofins.
  • the lyophilized pEX-K4-SQR was dissolved in lOmM Tris Buffer before using.
  • the pRSET expression vectors designed for high level protein expression and purification was used to perform SQR heterologous expression.
  • This plasmid is under control of the strong phage T7 promoter inducible using IPTG, present an (His) 6 tag at its N-terminal side and harbor a gene encoded a Blue fluorescent protein 'BFP' flanked between the enzymatic restriction sites of BamHI and Xhol.
  • the SQR synthetic gene of SEQ ID NO:6 was extracted from pEX-K4-SQR by double digestion using BamHI and Xhol restrictions enzymes and subcloned into pRSET A plasmid in place of the BFP gene to introduce a N terminal (His) 6 tag to facilitate purification steps.
  • the resulting plasmid pRSET- ABFP-SQR was used to transform E. coli C41(DE3) cells to ampicillin resistance.
  • the sequence of the Flis-tagged SQR of Aquifex aeolicus in pRSET T7 expression vector is provided in SEQ ID NO:2.
  • the pHisl7 expression vector was used to perform SQR Strep-tagged heterologous expression. This plasmid is under control of the strong phage T7 promoter inducible using IPTG.
  • a synthetic SQR gene (from Biomatik) with a double Strep Tag at its N-terminal side was cloned between the enzymatic restriction sites of Ndel and EcoRl.
  • the sequence encoding the N-terminal Strep-tagged SQR of Aquifex aeolicus is provided in SEQ ID NO:4.
  • the encoded protein is shown in SEQ ID NO:3.
  • Heterologous expression and purification A lOmL pre-culture of E. coli C45(DE3) cells was conducted by overnight at 37°C in 2xYT medium containing ampicillin (100pg/ml). This starter culture was used to inoculate 1L of culture performed at 37°C. Induction of SQR expression was conducted at an Aeoo of 0.4-0.6 by adding Isopropyl b-D- thiogalactopyranoside (500 mM final concentration). Cells were further grown at 37°C for 20h under 230 rpm agitation.
  • Cells were harvested by centrifugation (6 000 g for 30 min), resuspended in 40 mL of 20 mM Tris- HCI buffer (pH 8.0) and broken twice through a cell disruptor at 2.4 kbar in presence of proteases inhibitors mix (Halt protease inhibitor cocktail, EDTA-Free, Thermo Scientific). After low-speed centrifugation (10 000 g for 30 min), the membranes were collected by ultracentrifugation of the 10 000 g supernatant at 100 000 g for lh30. Membrane pellet was resuspended in 2 mL of 20 mM Tris-HCI (pH 8.0).
  • the membrane proteins (15 mg) were solubilized with 2% n-Dodecyl- -D-Maltopyranoside (DDM) in 20 mM Tris-HCI pH 8.0, 250 mM sodium Chloride (NaCI) and 10% glycerol. The suspension was incubated at 4°C on a shaker for 1 hour and then ultracentrifugated 30 minutes at 100 000 g. The supernatant was collected and diluted twice in the same buffer but without DDM to reduce DDM concentration to 1% before loading on the affinity resin.
  • DDM n-Dodecyl- -D-Maltopyranoside
  • the column was previously equilibrated with 20 mM Tris-HCI buffer (pH 8.0) containing 250 mM NaCI and 0.03% DDM.
  • the column was washed with increasing concentration of imidazole (5 mM, 25 mM and 50 mM).
  • SQR was eluted with 250 mM imidazole. Imidazole was removed by gel filtration equilibrated with 20mM Tris-HCI buffer (pH 8.0) containing 250 mM NaCI and 0.03% DDM.
  • Fluorescence spectra were recorded in a fluorescence spectrophotometer F-2500 from Hitachi. The sample was excited at a wavelength of 365 nm and emission was recorded at 480-600nm.
  • Exogenous Flavin Adenine Dinucleotide 'FAD' insertion Additional amounts of exogenous FAD were added in excess (4:1) between FAD and purified SQR.
  • the suspension was incubated for 2 hours at 37°C with gentle mixing. SQR was stored and used with excess of FAD.
  • Assays were conducted under anaerobic conditions at 40°C using a cuvette with a screw-cap equipped with a TeflonTM-silicon membrane purchased from Flellma Analytics.
  • Stock solutions of hydrogen sulfide (Na S) were prepared in 1 M Tris-HCI buffer (pH 8) and were frozen.
  • Fresh solutions of 40 mM Na S were prepared in milliQ water using the 1 M stock before activity assays.
  • Enzymatic activity was then monitored for 400 s by following the reduction of quinone at 278 nm minus 320 nm.
  • An extinction coefficient of 12 400 M ⁇ cm 1 (Lencina AM et al, 2013) was used to determine the extent of reduction of dUQ.
  • the slopes of the experimental curves were corrected by subtracting the slope of the curve before addition of SQR.
  • the V max and the Michaelis constant (Km) for dUQ were determined with the corrected slopes of the experimental curves and the Michaelis-Menten equation using GraphPad Prism (GraphPad Software, San Diego, CA).
  • the activity of immobilized SQR was first assessed using spectrophotometry.
  • the SQR-lpm beads fresh or lyophilized; 70 pg.mL 1 containing 1.5 pg.mL 1 ) were incubated at 40°C in 1.5 mL tubes containing Tris-HCI buffer 50mM, 0.03% DDM and 75pM of dUQand the initial absorption spectrum of the solution was measured.
  • Enzymatic reaction started by adding 200 pM of Na S to the reaction tube. After 5 min, the tubes were placed for 10 minutes on a magnetic stand to collect the beads on the side. The supernatant was taken out and final absorption spectrum was recorded. Control spectra were recorded with freshly washed magnetic beads without SQR incubation step.
  • the cosubstrate CoQl (40 pM) was added and a control cyclic voltammetry experiment was performed at 20 mV.s 1 from -0.5 to +0.5V versus a Ag/AgCI reference electrode.
  • Typical chronoamperometric experiments were performed by applying a fixed potential of 0.3 V for 1200 s. After stabilization of the current intensity at a low level (between 200 to 500 s after the potential step, i ⁇ 1 nA), increasing concentrations of Na S (0.1, 0.5, 1, 5, 10, 50 pM) were added into the solution every 100 s from dilutions of the 1M solution (4, 20, 40, 200, 4000, 20 000 pM).
  • a selectivity test was performed using cysteine or glutathione instead of Na S as source of sulfide. The same results were obtained when streptavidin beads of 100 nm are used (not shown). The device was also tested in presence of biological samples (liver homogenate and plasma from rat) diluted 4 times in Tris-HCI buffer. The system was eventually tested in the presence of 100 pg of beads and it worked (data not shown).
  • Anaerobic conditions were performed using an oxygen removal system as described in Plumere et al (2012).
  • This system consists of a glucose oxidase 'Gox' enzyme type VII from Aspergillus niger (2000 U), glucose (50 mM) as substrate for this latter and a catalase 'CAT' from bovine liver (15 U) for dismutation of hydrogen peroxide.
  • the efficiency of oxygen removal was tested by monitoring the cathodic oxygen reduction signals after swinging the electrode potential between +0.4 and -1.4 V versus Ag/AgCI (not shown).
  • Reaction was initiated by the injection of sodium sulfide solution into the reaction mixture containing in addition to the oxygen removal system, 3.34 pM of purified SQR (156 pg) with exogenous FAD. Cyclic voltammetry (CV) was performed at 20 mV.s 1 from -500 to +500mV vs SCE and the electrolyte volume was lmL.
  • Aguifex aeolicus SQR enzyme was overexpressed in E. coli C45(DE3), a derivative of BL21(DE3) selected according to Miroux and Walker (1996), by using a synthetic version of the gene encoding this enzyme and was purified in two steps using affinity chromatography containing Ni-NTA metal and desalting column for the His-tagged construction, using streptavidin column and PD-10 for Strep-tagged construction.
  • the pRSET-SQR expression vector was transformed in C45(DE3) host.
  • Cells were broken by cell disruption and membranes (1.2 mg/ml) were solubilized with DDM (2%). Solubilized material was loaded on NiNTA column then on PD-10 desalting column (GE) to remove imidazole.
  • Characterization of the purified enzyme by using UV-visible spectrum shows the typical FAD absorption bands at 356 and 451 nm in the air oxidized sample. Flowever, the absorption spectrum revealed that up to 25% of the SQR protein monomer contain FAD cofactor. To increase the amount of FAD-bound SQR, the enzyme was incubated with an excess amount of exogenous FAD. Unbounded FAD remained in the enzyme solution, however it was verified that the remaining free FAD did not affect the enzyme activity. UV-stain free gel showed that the FAD-bound SQR had increased up to 80%.
  • the enzymatic activity of the SQR enzyme was measured in the presence of dUQ as a cosubstrate by monitoring the decrease of its absorption at 278 nm after addition of sodium sulfide solution using UV/visible spectrophotometer (figure 1A, B). Data were fitted with Michaelis-Menten curve to determine the kinetic parameters (Vmax, Km) of the enzyme, SQR His and Strep-tagged, for dUQ (figure 1C, D respectively).
  • the constant affinity (Km) for dUQ of his and strep-tagged SQR was in the micromolar range: 18 and 17 mM respectively to be compared with 2 pM for the native SQR according to Marcia et al. (2010).
  • the SQR enzyme catalyzes the oxidation of hydrogen sulfide and the reduction of membrane quinone and can theoretically be studied using electrochemistry. As there was no prior electrochemical studies involving this enzyme, it was first necessary to identify a suitable cosubstrate that can play the role of a shuttle between SQR and the working electrode.
  • cosubstrates including one or two-electron mediators.
  • the cosubstrate needs to be sufficiently hydrophobic to interact with SQR and sufficiently hydrosoluble otherwise it could adsorb on the carbon working electrode and therefore cannot diffuse in solution to ensure the shuttle of electrons exchange between the enzyme and the working electrode.
  • Coenzyme Q1 CoQl
  • dUQ just one isoprenyl subunit instead of 10
  • the solution of SQR-lpm beads was loaded in a flat microwell at the bottom of which was present two carbon-based screen-printed electrodes, i.e. a working electrode forming a circular disk centered to bottom of the microwell and a counter electrode surrounding the working disk electrode.
  • a small commercial Ag/AgCI electrode is added into the solution as a reference electrode.
  • the magnetic microbeads of the SQR-beads solution are brown.
  • the 1 pm microbeads were rapidly collected over the working disk electrode surface as attested by the supernatant solution which became transparent and the carbon disk electrode which turned brown ( Figure 4).
  • Searcy DG Searcy DG, Peterson MA. Hydrogen sulfide consumption measured at low steady state concentrations using a sulfidostat. Anal Biochem. 2004 Jan 15;324(2):269-75.
  • Electrochemical biosensors recommended definitions and classification, Pure Appl. Chem. Vol.71, N°12, pp.2333-2348, 1999.

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Abstract

La présente invention concerne un biocapteur électrochimique à base d'enzyme qui peut être utilisé pour détecter des niveaux spécifiquement faibles de sulfure d'hydrogène (H2S) dans des fluides biologiques. Ce biocapteur comprend généralement un système d'électrode et une enzyme de sulfure de quinone réductase (SQR) fonctionnelle. La présente invention concerne également des procédés et des systèmes de mesure de H2S dans des fluides biologiques au moyen de ce capteur.
PCT/EP2019/063872 2018-05-31 2019-05-28 Capteur bioenzymatique électrochimique pour mesurer le h2s dans des fluides biologiques Ceased WO2019229086A1 (fr)

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WO2014138999A1 (fr) 2013-03-14 2014-09-18 University Of Ottawa Procédés de diagnostic et de traitement de maladie intestinale inflammatoire
WO2015109221A1 (fr) 2014-01-16 2015-07-23 Calysta, Inc. Compositions et procédés de récupération d'huile et de gaz délaissés
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