GB2029013A

GB2029013A - A method of and a device for chemical analysis

Info

Publication number: GB2029013A
Application number: GB7919001A
Authority: GB
Original assignee: Dr Eduard Fresenius Chemisch Pharmazeutische Industrie KG; Dr Eduard Fresenius Chemisch Pharmazeutische Industrie KG Apparatebau KG
Current assignee: Dr Eduard Fresenius Chemisch Pharmazeutische Industrie KG; Dr Eduard Fresenius Chemisch Pharmazeutische Industrie KG Apparatebau KG
Priority date: 1978-06-29
Filing date: 1979-05-31
Publication date: 1980-03-12
Also published as: GB2029013B; FR2432047A1; DE2828525A1; JPS5533688A

Abstract

A method of chemical analysis comprises converting a substance catalytically with an enzyme by applying the substance in liquid, dissolved or suspended form to one side of a semipermeable membrane at the other side of which is located at least one enzyme, and detecting the reaction product. The invention also includes a device suitable for carrying out the method.

Description

SPECIFICATION A method of and a device for chemical analysis The invention relates to a method of and a device for chemical analysis.

In biochemical analysis, enzymes are frequently used in order to quantitatively determine the presence of substances the conversion of which is catalyzed in highly selective manner by the appropriate enzyme. This takes place usually by adding the enzyme to the substance mixture to be analyzed in order to obtain an easily detected reaction product which can subsequently be quantitatively evaluated, for example using a colour reaction or other known methods.

When using this method, however, a certain quantity of enzyme is consumed each time. In order to avoid this disadvantage, enzymes have been developed in an immobilized form, for example bonded to suitable polymers and thus rendered insoluble. This immobilized form makes it possible to use the enzymes permanently or at least over a certain period of time. Generally a plurality of sample conversions can be carried out successively with a reaction cartridge which contains these immobilized enzymes. In practical, applications however, there are disadvantages here too since the immobilized enzymes can become unusable relatively rapidly because of contact with the measuring medium, if this medium contains certain high-molecular admixtures such as protein substances. Moreover only a few enzymes are available in immobilized form so that this method is not generally applicable.Furthermore there is a restriction in that the immobilized enzymes have an activity which is drastically reduced as compared to the corresponding soluble form of the enzyme.

According to a first aspect of the invention there is provided a method of chemical analysis comprising converting a substance catalytically with an enzyme and detecting the reaction product, wherein the substance in liquid, dissolved or suspended form is supplied to one side of a semipermeable membrane at the other side of which is located at least one enzyme.

According to a second aspect of the invention there is provided a device for carrying out a chemical analysis wherein a reaction chamber is provided with a semipermeable membrane which forms the separating wall between two halves of the reaction chamber.

The invention makes it possible to use enzymes in the normal soluble form for the purposes described in an unlimited manner over a long period of time so that the above disadvantages are no longer present. The method in accordance with the invention and the corresponding devices may, on the other hand, be used to determine enzyme activity in a test medium. The invention is moreover suited to substantially prolonging the period of usability of the enzymes where immobilized enzymes are used.

The invention will now be described in greater detail, by way of example, with reference to the drawing, the single figure of which shows diagrammatically an analysis device in accordance with invention.

The arrangement shown comprises a chamber 1 a, 1 b, which is constructed from a material which is neutral to the reagents in question and is subdivided into two halves by a semipermeable membrane 2. The medium to be analysed passes through first half 1 a of the chamber the medium flowing in through a line 3 and out through a line 4.

The second half 1 b of the chamber is filled with the enzyme, for example, in the form of an enzyme solution. Filling of the chamber part 1 b with enzyme solution takes place through a line 5 while a line 6 serves for venting. Valves 7 and 8 serve to block off these two flow paths after filling.

The membrane 2 is permeable to the lowmolecular portion of the substance of the medium to be analysed, this portion being the part which is of interest, but the membrane 2 is impermeable to the enzyme. Because of the high molecuiar weight of the enzymes (X1 00,000 is typical) a suitable membrane can be easily found. For example, the membrane may comprise a cellulose derivative, polyacrylonitrile, polysulphone or polycarbonate.

Portions of the measuring substance consequently diffuse out of the half 1 a of the chamber through the membrane 2 and into the chamber half 1 b and are caused to react there by the enzyme. The reaction products which are also of low molecular size diffuse partially back into chamber half 1 a. A flow balance arises and results in differences in concentration, both of the components involved in the reaction and of the reaction products, between the concentration in the supply line 3 and the concentration in the outlet line 4 of the chamber half 1 a. After corresponding calibration, as the starting concentration of one of the reactants can be deduced by measuring one of the reaction components or one of the reaction products at the outlet and comparison with the corresponding value at the inlet.Thus two measuring sensors 9 and 10 are provided in the drawing and are connected to a measuring device 11.

The chamber half 1 b is charged with the enzyme glucose oxides for example, which has the property of converting glucose into gluconic acid by consuming oxygen, in order to determine the glucose content in a blood serum. By measuring the oxygen content at the inlet and at the outlet with suitable measuring sensors 9 and 10 the consumption of oxygen can be determined and thus conclusions can be drawn with respect to the original glucose content of the serum. The measuring sensor 9 may be dispensed with if the starting oxygen content of the measuring medium is sufficiently accurately known.

Another example of analysis consists in determining the urea content of a measuring solution. Urease is suitable as an enzyme in this case. It converts urea and splits up urea while releasing ammonium ions. Ammonium ionselective electrodes may be used in this case as the measuring sensors 9 and 10.

The measuring sensors 9 and 10 can operate basically in accordance with the various known measurement principles. Besides the gas-sensitive and ion-selective electrodes already mentioned, enzyme electrodes, thermal measuring sensors and, in particular, photometric measuring devices may be used. Certain errors can, in addition, be greatly reduced by applying a differential measuring technique in some circumstances in conjunction with a reversal of the flow direction in the chamber half 1 a or by exchanging the two measuring sensors 9 and 10 for two successive measurement of the same sample.

With certain measurements, it may be advisable or necessary to add indicators to the measuring material, which indicators are either involved in the enzymatic reaction as reactants or permit simple quantitive determination by means of an auxiliary reaction with one or more of the reaction products. An example of this is adding colour indicators in photometric evaluation.

Moreover, it may be advisable to add buffer solutions to the measuring material in order to keep the enzyme activity within an optimum range.

In certain cases, a multi-stage enzymatic conversion is necessary in order to obtain a quantitatively and easily detected reaction product.

In this case, the chamber half 1 b may contain a mixture of two or more enzymes. Alternatively, or in combination therewith, the outlet 4 of the arrangement shown in the drawing may be combined with the inlet of an arrangement, formed in the same manner, in order to carry out a further conversion with another enzyme in the second arrangement.

When using immobilized enzymes instead of soluble enzymes, -the described arrangement has the advantage as compared with direct utilization of immobilized enzymes that contamination with high-molecular components of the -measuring material is avoided because the semipermeable membrane 2 prevents the passage of these substances. As a result, the duration of the usefulness of the immobilized enzyme can be extended substantially.

The device shown schematically in the drawing is, on the other hand, also suitable for determining enzyme activity in a measuring solution. For this purpose the chamber half 1 b is charged with the measuring solution. A normal solution flows through the chamber half 1 a and contains a substrate of the relevant enzyme and possibly other necessary reaction components in a predetermined concentration and possibly also indicators, buffers etc. Because of the display obtained with the aid of the measuring sensors 9 and 10, conclusions can then be drawn as to conversion of the substrate and thus with regard to the enzyme activity.

Claims

1. A method of chemical analysis comprising converting a substance catalytically with an enzyme and detecting the reaction product wherein the substance in liquid, dissolved or suspended form is supplied to one side of a semipermeable membrane at the other side of which is located at least one enzyme.

2. A method according to claim 1, wherein the enzyme is dissolved or suspended in a liquid.

3. A method according to claim 2, wherein the carrier liquid of the enzyme is supplied continuously to the said other side of the membrane.

4. A method according to claim 1, 2 or 3 wherein the enzyme is immobilized.

5. A method according to any one of the preceding claims, the enzyme consumed is ascertained by differential measurement.

6. A method according to any one of the preceding claims, wherein a buffer solution is supplied to the material to be measured in order to keep the enzyme activity within an optimum range.

7. A method according to-any one of the preceding claims wherein two or more enzymes are supplied at the same time or successively to the said other side of membrane.

8. A device for carrying out a chemical analysis wherein a reaction chamber is provided with a semipermeable membrane which forms the separating wall between two halves of the reaction chamber.

9. A device according to claim 8, wherein detection devices are provided for detecting the consumption of enzyme and/or the consumption of substrate.

1 q. A method of carrying out chemical analysis substantially as described herein with reference to the drawing.

11. A dev.ice for carrying out chemical analysis substantially as described herein with reference to the drawing.