A METHOD AND APPARATUS FOR INDICATING THE PRESENCE OF SUBSTANCES WHICH,IN A CHEMICAL REACTION,GENERATE OR CONSUME GAS

Abstract: Method of indicating the presence of a detectable gas in gas-generating or gas-consuming reactions, by the use of a hydrogen and ammonia sensitive, palladium coated semiconductor device (Pd-MOS-FET). The measurements are carried out in the gas phase in a gas detector which is disposed in association with the reaction vessel in which the reaction which is to be studied takes place. A particular study has been made of enzymatic reactions in which hydrogen and/or ammonia are generated by the action of the enzyme urease on urea and creatininase on creatinine.

Description: A METHOD AND APPARATUS FOR INDICATING THE PRESENCE OF SUBSTANCES WHICH, IN A CHEMICAL REACTION, GENERATE OR CONSUME GAS The present invention relates to a method and an apparatus for indicating the presence of substances which, in a chemical reaction, generate or consume detectable gas. It is known in this art that an ion-selective elec¬ trode in combination with a gas-permeable membrane can be used for measuring the amount of ammonia which is generated in certain enzymatic reactions. In this case, the electrode is kept in a buffer solution. It is also known that an apparatus having an ion-selective elec¬ trode placed above a thin air gap in a reaction vessel may be used. In these prior art cases, the concentra¬ tion of a certain ion is measured, and not the generated gas itself. These prior art methods are not particularly sensitive or rapid, and they restrict the number of gases or reactions which can be studied. A new method and a new apparatus for indicating the presence of substances which, in a chemical reaction, generate or consume detectable gas have now been developed, these constituting the subject matter of the present invention. In the method according to the present invention, those substances whose presence is to be indicated are caused to react for the generation or consumption of detectable gas, whereafter the detectable gas, if such is present, is caused to pass a gas detector which is selectively responsive to the detectable gas. This measurement method is not electrochemical, but is characterized in that the generated (or rest of the consumed) gas is detected by means of a catalytically active detector in the gas phase. As a result, a greater degree of flexibility will be attained as regards the number of gases (the number of reactions) which can be studied, and, in many cases,, a superior level of sensitivity and rapidity in relation to that which can be achieved using prior art methods. The present invention also relates to an apparatus for carrying out the novel method. This apparatus comprises a reaction vessel and a gas detector disposed in conjunction therewith, the detector being sensitive to the detectable gas. In a particularly preferred embodiment of the method according to the invention, an enzyme is caused to act on a carrier for the enzyme, a gas, such as, for example, hydrogen, ammonia or hydrogen sulfide, bein generated. This gas is then caused to pass a detector which is sensitive precisely to the gas in question. Suitable gas detectors are so-called Pd-MOS-structures which are responsive to hydrogen-containing gases, but other gas detectors may also be used, such as for example a (warm) platinum thread, a semiconducting metal oxide (ZnO, Sn0 2 etc) , or an oscillating piezoelectric crystal. H 2 9 2 can also be detected by means of the apparatus according to the present invention. Furthermore, the method andapparatus according to the present invention may be applied to a semiconductor sensor based on silicon, the current strength of the sensor changing in the presence of, for example, ammonia In certain cases, it may be advisable to have a carrier gas (i.e. air) flow through the reaction vessel. This is particularly favorable when the invention is used to indicate the consumption of a gas in a reaction. In such an instance, a minor amount of the gas which is being consumed is introduced into a carrier gas which, after the reaction, is caused to pass the detector which then determines the amount of remaining reacting gas in the carrier gas. O P In certain cases, it may also be necessary to decompose the gas in question before it reaches the sensor element in the detector. Such a decomposition stage is considered as forming part of the concept of a gas detector. The gas detector may be disposed directly above the surface of a solution in which, for example, enzyme and carrier are allowed to react. The detector then responds to the gas which is released in the enzymatic reaction and is dissipated out into the air gap above the solution. In another embodiment of a present invention, an immobilized enzyme is caused to react on a carrier in a through flow cell, in which the detector is disposed in an air gap above the liquid flow. Furthermore, an immobilized enzyme may be disposed closely.adjacent the detector, for example by being chemically coupled to the detector. The apparatus and the method according to the present invention may apart from in enzymatic reactions, also be used in reactions in which are included micro¬ organisms which generate or consume gases. Of particular interest in this context are microorganisms which develop hydrogen gas and methane gas. The nature of the present invention in its aspect will be more readily understood from the following brief description of the accompanying drawings, and discussion relating thereto. In the accompanying drawings: Figs. 1-3 show three different embodiments of the apparatus according to the present invention, also called an enzyme transistor ? . Fig 4A and B schematically show illustrations of hydrogen-sensitive detectors; Fig 5 shows a calibrating diagram for ammonia obtained with a detector according to Fig 4; 4 Fig 6 shows a curve of the response of the detector to a reaction in which urea is hydrolized by means of urease; Fig 7 illustrates the hydrogen gas production from 5 Clostridium aceto butylicum as a function of time.; Fig 8 shows a standard curve for NADH; and Fig 9 shows a standard curve for NAD . More precisely. Fig 1 illustrates an apparatus comprising a gas-sensitive detector disposed above the 10 surface of a solution in which an enzyme and a carrier- therefor are located. Here, the detector responds to the gas which is released in the enzymatic reaction and which is diffused out into the air gap above the solutio Fig 2 shows an apparatus in which a carrier gas is 15 allowed to pass a solution with reactants, whereafter the carrier gas, entraining gas from the reaction, passes a gas detector. This apparatus is also suitable for use when it is desirable to study a gas-consuming reaction, a minor amount of the gas which is to react 20 being introduced into the carrier gas. The amount of remaining gas in the carrier gas is thereafter measured by the gas detector. Fig 3 illustrates a through-flow cell for reaction between an immobilized enzyme and a carrier in which 25 the detector is disposed above the liquid flow in some part of the cell. The invention will now be described in greater detail below by means of the following examples which are not intended to restrict the spirit and scope of 30 the present invention as are apparatent from the appende claims. EXAMPLE . 1 The reaction between urea and urease, during which ammonia is generated, was studied. As gas detector, use 35 was made of a hydrogen and ammonia sensitive Pd-MOS- OMPI -structure . These hydrogen-sensitive Pd-MOS-structures have been described, int al, in Swedish patent specifi¬ cation 7411342-4. Fig 4 schematically illustrates two embodiments of these structures. In the first instance, Fig 4A, hydrogen-containing gases (H-, H 3 , H 2 S etc) realize a shift of the capacitance-voltage curve along the voltage axis, and in the second case, Fig. 4B, a shift of the current-voltage curve along the voltage axis. This shift, which may be of the order of magnitude of 0.5 V, may readily be measured. This type of detector has a response, shown in Fig. 5, to ammonia, which has been generated by the reaction between NH.C1 and NaOH in an apparatus according to Fig. 1. EXAMPLE 2 Urease (0.5 I.U) was added to a solution containing urea (1 M urea in 1 ml 0.1 M Tris-HCl. pH 8.5). After 6 min., the reaction was stopped by the addition of HgCl„ . The response from the hydrogen-sensitive Pd-MOS- -structure in an apparatus according to Fig 1, is shown in Fig 6. The slope of the curve (the broken line in Fig 6) may be used to determine the amount of urea which was present in the measurement cell. This slope is unambiguously determined by the urea concentration. Urea concentrations of as little as 0.1 mM may easily be determined in this manner. No attempt at optimation was made in the experiment, for which reason it is probable that a much higher degree of sensitivity can be attained. EXAMPLE 3 The signal from a Pd-MOS-structure was compared with the signal from an enzyme transistor in which the Pd-MOS-structure was used as a gas sensor in an apparatus according to Fig 3. The reaction which was studied was deimination of creatinine by means of immobilized creatininase. The correlation between the signals from the enzyme transistor and the gas detector was very good. O PI EXAMPLE 4 A Pd-MOS-capacitance provided as gas sensor accor¬ ding to Fig 3 was used continuously to follow the H 2 generation from a 5 ml tower packed with immobilized (on balls of alginate gel) Clostridium acetobutylicum under anaerobic conditions. The buffer flow through the tower was 0.3 ml/min and generated gas was diluted 10 times prior to analysis. The thus obtained H~ generation is illustrated in Fig 7. Finally, mention might be made of a further interes ting field of application of the present invention, namely the use of the enzyme hydrogen dehydrogenase (E.C.I.12.1.2) which provides the possibility to study reactions in which hydrogen and the coenzymes NAD(P) participate. EXAMPLE 5 Fig 8 illustrates a standard curve for NADH, in which 2.5 ml samples of varying concentrations were introduced into the buffer flow. The tower contained immobilized hydrogen dehydrogenase of a volume of 1 ml. The amount of generated hydrogen gas is expressed as ppm in the carrier gas. Fig 9 illustrates a standard curve for NAD , in which 2.5 ml samples of varying concentrations were introduced into the buffer flow. The tower contained immobilized hydrogen dehydrogenase of a volume of 1 ml. The amount of hydrogen gas present in the buffer corres¬ ponds to 100 ppm in the carrier gas flow. OUP

Claims: CLAIMS 1. A method of indicating the presence of substances which, in a chemical reaction, generate or consume detectable gas, wherein the substances are caused to react for the generation or consumption of detectable gas, whereafter the detectable gas, if such a present, is caused to pass a gas detector which is selectively sensitive to the detectable gas. 2. The method as recited in claim 1, wherein the reaction is carried out in a solution, and wherein the gas is caused to pass a gas detector placed above the surface of the solution. 3. The method as recited in claim 1, wherein the reaction is carried out in a through flow cell, the gas detector being placed in the outlet from the cell. 4. The method as recited in one or' ore of the preceding claims, wherein a carrier gas flow is used to lead the detectable gas from the reaction to the gas detector. 5. The method as recited in one or more of the preceding claims, wherein the detectable gas is hydrogen, ammonia, hydrogen sulfide or orther dissociable, hydro¬ gen-containing gas. 6. The method as recited in one or more of the preceding claims, wherein use is made of a Pd-MOS-struc- ture as gas detector. 7. The method as recited in one or more of the preceding claims, wherein the reaction takes place in the presence of an enzyme, such as urease, creatininase or hydrogen dehydrogenase. 8. The method as recited in one or more of the preceding claims, wherein the detectable gas is decomposed before being caused to pass the gas detector. 9. An apparatus for indicating the presence of substances which,, in a chemical reaction, generate or consume detectable gas, comprising a reaction vessel and a gas detector disposed in association therewith, the detector being sensitive to the detectable gas. 10. The apparatus as recited in claim 9, wherein the gas detector is disposed above the surface of a solution in which the reaction is carried out, in the outlet of a throughflow cell, or in a carrier gas flow which has passed the reaction vessel. 11. The apparatus as recited in claim 9 or 10, wherein the gas detector is a Pd-MOS-structure.

Inventors: DANIELSSON B, LUNDSTROEM I, MOSBACH K, STIBLERT L
Applicants: DANIELSSON B, LUNDSTROEM I, MOSBACH K, STIBLERT L
Application year: 1980
Main classification: G01N27
IPC codes: C12M1, C12Q1, G01N27