DK146399B - PROCEDURE FOR ENZYMATIC DETERMINATION OF OXIDIZABLE OR REDUCABLE SUBSTANCES AND APPARATUS FOR IMPLEMENTING THE PROCEDURE - Google Patents

Description

(19) DENMARK

| É (12, PRESENTATION ο „146399 Β

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Patent Application No: 3032/72 (51) Int.CI.3: G01N 31/14 (22) Filing Date: Jun 16, 1972 (41) Aim. available: 19 Dec 1972 (44) Submitted: 26 APE 1983 (86) International Application No: - (30) Priority: 16 Jun 1971 DE 2130340 (71) Applicant: 'BOEHRINGER MANNHEIM GMBH; 68 Mannhelm-Waldhof, DE.

(72) Inventor: Alexander 'Hagen; DE, Wolfgang 'Gruber; DE, Hans Ulrich 'Bergmeyer; DE, Dieter Maworek;

THE

(74) Clerk: Chaa. Hude_ (54) Process for enzymatic determination of oxidizable or reducible substances and apparatus for carrying out the process

The invention relates to a method for quantitatively determining substances in an oxygen-containing aqueous solution by oxidation or reduction in the presence of an oxygen-transferring carrier-bound enzyme in which an aqueous solution of the substance to be determined is introduced into a constant-flow liquid stream of a buffer solution having a specific oxygen concentration, after which the solution is passed over the carrier-bound enzyme and the change in the oxygen concentration is measured by an oxygen-sensitive electrode.

Substrates of oxygen-transmitting enzymes are continuously being quantitatively determined in the art and in the clinical laboratory to a large extent. Here, there is generally the problem of implementing these provisions quickly and simply, ie. without the use of special purification and separation methods, so that the substance to be determined is chemically converted into a quantitatively measurable product.

2 146399

Enzymatic processes are frequently used here. Thus, for example, D-glucose is oxidized by the enzyme glucose-oxidase with the participation of molecular oxygen to gluconic acid. The hydrogen peroxide thus produced at the same time is reacted with a leuko dye to form a dye whose extinction is determined in the photometer and which is equivalent to the amount of glucose. This process, which is a typical example of many substrate determinations, for example of uric acid, galactose and the like, has numerous disadvantages.

In general, biological tests have to remove the protein, which means an additional working step. The method requires a relatively large amount of time, so that the sample rate for serial analyzes can only be large where very expensive analytical machines are used.

Therefore, for blood glucose testing, devices were developed early that use oxygen determination as a measurement principle and therefore do not exhibit some of the disadvantages mentioned. Here, the oxygen content of the solution is measured by means of an oxygen sensitive electrode. The glucose oxidase added causes oxygen oxidation of glucose during consumption. The change in oxygen concentration is measured with the electrode and is proportional to the amount of glucose reacted. This eliminates the need for protein removal in the samples, shortening the time between sample application and the measurement result realization, and therefore the accuracy of the specific analysis becomes high due to the absence of connected laser-specific indicator reactions.

However, this approach also has numerous disadvantages. Thus, the volume of the sample to be determined must be determined by means of adjusted measuring pipettes, which not only mean labor consumption but also provide significant error possibilities. Thus, methods which operate discontinuously are known. Here, the oxygen-sensitive electrode containing the measuring cell must be filled with a certain amount of reagent. A disadvantage here is the possibility of formation of air bubbles at the electrode surface, which disturbs the determination.

A defined sample quantity must be introduced into the stirred solution and after a specified time a measurement value must be read. Before carrying out a new measurement, the cell must be emptied. If the system has not been in operation for some hours, a lengthy and long-lasting adjustment is necessary, which is probably due to changes at the surface of the electrode.

3 146399

Therefore, a method has already been described in which an oxygen sensitive electrode is still flowed by a reagent containing glucose oxidase. The sample application is made here from a secondary stream containing the glucose to be determined, glucose being dialyzed in the reagent stream through a semipermeable membrane. This method has the disadvantage that the time until a measurement result is available is relatively long as the dialysis proceeds slowly. Still, a method is known in which immobilized glucose oxidase is placed immediately at the electrode surface. The oxidation of the glucose occurs at the electrode surface.

In practice, this method is only suitable for discontinuous glucose determination, since only limited amounts of enzyme can be fixed at the surface of the electrode, so that the glucose turnover is relatively long.

The invention, therefore, is to provide a method which avoids the disadvantages described and, in particular, provides a fast, error-free and at all times operational method for carrying out even larger determinations.

According to the invention, this task is solved by a method characterized by administering a certain amount of the solution containing the substance to be determined, air-blister-free in a constant-flow liquid stream of a buffer solution of a certain oxygen concentration in a blocky manner without changing the flow rate, it passes over a carrier-bound enzyme and subsequently leads to the electrode.

Suitable for the process according to the invention are those substances which can be reacted in the presence of oxygen transferring enzymes during the uptake or delivery of oxygen. Examples of such substances are sugars such as glucose and galactose, uric acid, nucleoside bases such as xanthine and hypoxanthine, cytochrome, amino acids, polyunsaturated fatty acids, hydrogen peroxide and inorganic peroxides.

if 148399

As oxygen-transferring enzymes which, within the scope of the invention, bound to one carrier can be added, mention is made of glucose oxidase, galactose oxidase, uricase, xanthine oxidase, cytochrome oxidase, amino acid oxidases, lipoxygenase, peroxidase and catalase.

The oxygen-transferring enzymes must be bound to a carrier within the scope of the process of the invention. Suitably, the carrier must be such that the enzyme binds to highly specific activity and that destruction of the carrier and loss of enzyme activity are largely excluded. A suitable carrier is described, for example, in German Publication No. 19 08 290.

Preferably, the carrier should be in particulate form suitable for column filling flowing through the buffer solution, thereby eliciting some mixing of the sample volume in the buffer solution block-wise or propagely with the buffer solution. A highly specific activity of the carrier-bound enzyme is desirable as this enables the use of very small amounts of carrier-bound enzyme, thereby lowering the mixing of the sample "block" with the buffer solution and increasing the sample frequency and obtaining readily and quickly usable measurement results. the specific activity of the carrier-bound enzyme used is at least 10 U / g, preferably more than 50 U / g and more preferably more than 100 U / g.

An essential feature of the method according to the invention consists in the air-blister-free, block-like introduction of the solution containing the substance to be determined (for example, the glucose) into the buffer solution stream. The solution must therefore be brought in as little mixing as possible with the buffer solution stream so that it migrates in the same way as a plug in the liquid stream. Such an introduction can be carried out, for example, by means of a slider which contains in a bore the measured amount of the solution which is introduced into the liquid buffer solution stream so that the sample solution is fed blocky or propagate into the buffer solution.

A further essential feature of the process according to the invention consists in the use of a constant flowing stream of a suitable buffer solution as a carrier and reaction agent for the reaction. Here, constant velocity is understood to mean such a constant in velocity that the inevitably occurring velocity variations are so small that they are not practically noticeable on the recording instrument associated with the electrode. This condition is ideally met when a constant oxygen concentration is recorded as long as no sample solution block passes the electrode. If this constant rate is not maintained, a change in the oxygen concentration will be possible with the still recording instrument. Such a change could also be avoided when introducing air bubbles into the reagent stream when introducing the sample solution.

Essentially, it is furthermore that the blocky introduction of the sample solution does not induce any change in the flow rate in the constant flow flow of the buffer solution, as there would also be a danger of a misinterpretation of the measurement result. First, the simultaneous fulfillment of all of the essential features of the method of the invention set forth above enables the advantages described below to be achieved and to create an ever-operating fully automatic working apparatus which performs the desired measurements, without any pipetting, volume measurements, time measurements or similar are needed.

Suitable oxygen sensitive electrodes for carrying out the method of the invention are well known. Examples of such suitable oxygen sensitive electrodes are Saurstoff-Electrode from L. Eschweiler und Co., Polarographic Oxygen Sensor 39 550 from Beckmann Instruments, Sauerstoff electrode from Hartmann und Braun AG, Frankfurt.

Preferably, the method is carried out so that, after passing the electrode, the buffer solution is circulated, again consuming components. For example, if the process is carried out to determine a substance which is oxidized during an oxygen consumption, the oxygen content of the buffer solution is again supplemented, for example, air being passed, which is conveniently done in a somewhat larger container at a suitable residence time of the buffer solution there. If the substance to be determined is reduced while increasing the oxygen content of the solution, the excess oxygen is again removed, correspondingly passing through an oxygen-free carrier gas, for example nitrogen or an inert gas.

The buffer solution itself consists of an aqueous solution of a suitable buffer for the enzyme used at each time.

The suitable buffers and buffer concentrations as well as the favorable pH values for the constantly used enzymes are known.

The buffer solution may optionally further contain other substances besides the buffering compounds necessary for the enzymatic reaction. Examples thereof are salts containing the necessary ions, such as, for example, magnesium ions, sulfate ions and the like, or stabilizing compounds, surfactant substances and the like.

The blocky introduction of the sample solution without changing the flow rate is preferably carried out such that the buffer solution stream is divided into two partial streams and the partial streams alternately interrupted, and a certain amount of the solution of the substance to be determined (sample solution) is introduced into one partial stream at the same time. interruption of the second subcurrent. It is particularly advantageous here to add the sample solution at the site of interruption itself.

Here, preferably, an excess of the sample solution is added, e.g. of serum, and when opening the sub-stream separated from the excess. This can be done, for example, as already indicated above, since a slider-like device is inserted in a partial flow of the buffer solution, whose slider has a bore adjusted for recording a certain amount of sample solution. By immersing this slider opening in the excess sample solution, e.g. the excess of sample serum, or by opening the bore to a larger container containing such a solution, and by allowing the sample to flow, a metered amount of sample solution upon displacement of the slider is separated and block-wise administered into the buffer solution stream as the bore of the slider is brought into the interrupted subcurrent of the buffer solution that at this bore a reopening of the interrupted flow occurs, Preferably simultaneously the second uninterrupted subcurrent interrupted, which can happen at the same slider.

An apparatus for carrying out the method according to the invention is characterized by a buffer reservoir, a sample applicator with at least two interlocking passageways alternating at least two corresponding bores, one bore of which communicates with a supply line for dissolving the substance. to be determined when the corresponding passageway is blocked, a constant-performance pump in the line in front of or behind the applicator, a column of carrier-bound enzyme, and a detector with an oxygen-sensitive electrode connected to an amplifier and a recording instrument interconnected with each other in this order by pipelines.

According to a particularly preferred embodiment, the applicator 1 is connected to a suppressor-providing device, for example a vacuum pump, with which the supply solution for the sample solution and the sample well in the slider can be evacuated.

In a preferred embodiment, the applicator consists of a sheath with a displaceable core, the sheath and core each having three passages which can be covered by displacement of the core. Particularly advantageous is such an applicator characterized by a casing having a first and a second inlet bore, a first and a second outlet bore, an air inlet bore, a vacuum bore and a sample solution bore, and a core which is displaceably disposed in the housing with three bores so arranged that by displacing the core, the first input bore can be connected to the first output bore, the second input bore connected to the second output bore, the air input bore connected to the vacuum bore, and the sample solution bore to the vacuum bore, the first input bore being unable to connect to the first output bore simultaneously with the second bore. connect to the second output bore.

For example, as a pump with constant output in the line before or behind the appliance, a hose pump can be used. A preferred one. hose pump is stated in German publication no.

20 25 056.

146399 δ

The method and apparatus according to the invention will be explained in more detail below with reference to the attached drawing. On this are: fig. 1 is a schematic representation of the apparatus according to the invention; FIG. 2 is a schematic representation of the conductivity of the buffer solution stream at the oxygen electrode; FIG. 3 is a sectional view of the column filled with the carrier-bound enzyme; FIG. b is a representation of an applicator according to the invention with connecting cables as well as in longitudinal section and cross section; 5 shows a registration curve; FIG. 6 an adjustment curve; FIG. 7 shows a further registration curve and FIG. 8 the corresponding adjustment curve.

As shown in FIG. 1, the reservoir R contains a buffer solution buffer. The pump P enters the buffer solution from the reservoir R into the sample applicator A and from there into the column S filled with the carrier-bound enzyme and further into the measuring electrode containing detector D and finally back to the reservoir. The detector is connected to an electronic amplifier V, which amplifies the measurement signals from the electrode and conducts them to record the recording instrument G, which may be, for example, a printer.

The reservoir R may consist of a suitable container which has a discharge opening for buffer solution. In the preferred embodiment of the method according to the invention with the buffer solution in circuit 9, it has an additional buffer solution inlet opening and suitably an opening for inlet air. In this embodiment, a pump M serves to generate an air flow. For example, here the pump used in aquariums is suitable. Preferably, the reservoir consists of a container of thermostated design. The pump P, which serves to conduct the buffer flow, may, as already mentioned, be designed as a hose pump or as a piston or turbine pump. Preferably, its performance is adjustable.

The FIG. 2 is a schematic sectional view of a detector cell 1, which is preferably thermostatically constructed. It contains an oxygen-sensitive electrode 2 as well as a solution channel 3? into which the measuring electrode protrudes.

The term "amplifier V" is intended to mean all functional parts which the measuring electrode needs to operate, such as a power source, power supply connections, electronic amplification, and so on.

The recording instrument G serves to record the measurement results. Various tools can be used, however, preferring a printer that can be connected to the amplifier V. Instead of the printer, other analog or digital instruments can be used. For the measurement it is only necessary to indicate the measurement result in relation to the time course. In many cases, simply recording the measurement signal maximum is also sufficient.

The FIG. Column 3, in section 3, consists of a column container k, which preferably has a cylindrical shape, which is closed at the ends and at one end has an inlet opening 5 and at the other end an outflow opening 6 for buffer solution. It is loaded with grain 7, which consists of enzyme bound on a solid insoluble carrier. The size of the column depends on the desired measurement sensitivity, the amount of measurement sample and the activity of the enzyme. Preferably, a column is used in thermostated design. Conveniently, the column is arranged so that it can be easily exchanged if the activity of the carrier-bound enzyme has become too low, or for some other reason an exchange is necessary, f.

For example, if the apparatus is to be adapted for the determination of another substance, another carrier-bound enzyme must therefore be introduced.

Sample applicator A serves as above for air-free, block-like introduction of the sample solution into the buffer stream. It results in the measurement of the sample solution and the continuous maintenance of the buffer flow without any large velocity variations.

In the embodiment of FIG. In the embodiment shown, the applicator consists of a casing 10 and a core 11 which is displaceable therein. An inlet opening 12 for the buffer flow leads to the bore 12a in the casing 10. A branch line 13 exits from the line 12 and leads to the bore 13a in the casing 10. · "Similarly passes an output line 1H, which is connected to the bore 1 '+ a, which serves to output the buffer solution, on the other side of the casing 10. A branch line 15 connects the output bore 15a of the casing 10 with the output line l * t. A conduit 16 communicating with a bore 16a in the casing 1Q is connected to a vacuum.

The displaceable core has bores 17, 18 and 19 which, at the corresponding slider position, can each time bore 12a and 1a + 1a, 13a and 15a as well as 16a with bore 20 or funnel bore 21 in casing 10.

Referring to FIG. From the normal position of the core 11 shown, the sample solution to be examined, for example serum, is introduced into the funnel-shaped opening .21 and thence into the bore 18 of the core 11. The filling height of the funnel-shaped bore 21 on the casing 10 does not matter here, when only the bore 18 in the core 11 is completely filled. As the sample is filled, the buffer current flows from 13a to 15a. At the end of the filling, the core is pushed in the direction of the arrow until the bore 18 covers with the bores 12a and 1a. The sample solution, the volume of which corresponds exactly to the volume of the bore 18 in the core 11, is introduced by the buffer stream block-wise into the column S. Yed. position of the core 11, the current is interrupted from 13a to 15a, and simultaneously. you, the amount of solution remaining in the filling opening 21 is aspirated at the bore 19, bore 16a and line 16. As soon as the sample solution is discharged into the buffer flow, the core 11 is pushed against the arrow direction until the bore 18 covers with the air inlet bore 20 and the exit bore 16a. The bore 18 is thereby emptied of buffer and the buffer flow from bore 13a to bore 15a is released again. Finally, the core is again moved in the direction of the arrow to the initial position and the process can be repeated.

The size of the applicator, in particular the volume of the bore 18, depends on the sensitivity of the method, the flow rate of the buffer solution and the size of the column S. If a bore 18 is desired for the bore 18, a corresponding opening can be placed in the edge 10 opposite the filling opening 21. The sample will not be able to run out, as the surface tension in the droop below will prevent a tear.

The applicator may consist of any material if this is inert to the solutions used and allows a dense core of core and sleeve. Suitable materials are for example glass, plastic or metal. The applicator may also consist of various materials, for example of metal with surfaces of a slip-resistant plastic, such as polytetrafluoroethylene.

The applicator may also be carried out in a different way within the framework of the above-mentioned principle, for example cylindrical in accordance with a multi-way cock. In this case, the parallel flow is suitably controlled in another tap whose core is on the same axis as the core in which the sample solution is applied.

The method and apparatus of the invention exhibit a number of particular advantages. They allow for extremely fast analyzes, since the measurement results are already available in a very short time (about 10 seconds to 1 minute). With the constant fluid flow, a very stable adjustment of the electrode can be ensured, which makes a long-lasting post-adjustment superfluous and which guarantees continuous operational readiness. The use of adjusted measuring pipettes becomes superfluous, eliminating the resulting errors. By using carrier-bound enzymes, another significant saving is also achieved, since the enzyme cannot be used only once, as with dissolved enzymes, but can often be used again. For example, an apparatus according to the invention with carrier-bound glucose oxidase has been in operation for more than six months, without the need for a replacement of the enzyme or a readjustment of the electrode.

12 146399

The following examples further illustrate the invention.

Example 1

Determination of D-glucose in serum with glucose oxidase according to the equation: Glucose + O2 + EGjO ^ lucoseo.xydase y gluconic acid + H ^ And

It was applied to the FIG. 1. Column S had a diameter of 1 cm and a filling height of 1 cm and was filled with water swollen glucose oxidase on carrier with a specific activity of 200 U / g.

As detector, one of the company W T W in Weilheim / Obb was used. Negotiated oxygen sensitive electrode with an oxygen measuring device negotiated by the same company designated Oxi 610 E and the connected potentiometer Ee 511 from Servogor.

The outflow rate in the electrode was 7? 5 cm / sec., The diameter of the ... entry bore in the flow cell shown in FIG. 2, was 1 mm.

In principle, the applicator used had the one shown in FIG. h. The bore 18 in the core 11 had a diameter of 2 mm and a length of 10 mm. The applicator consisted of glass.

The individual appliance parts were connected to 1 mm diameter silicone hoses. As buffer, 0.2 M potassium phosphate buffer of pH 6.0 was used, containing 18 mM potassium iodide, 7.5 mM ammonium heptamoyl lybdate and 800 mM sodium chloride.

In the applicator, serum samples with a specific glucose content were administered at a distance of one minute. Samples 1 to 3 each contained 100 mg glucose / 100 ml serum. The sample ^ contained 200, the sample 5,300, the sample 6 ^ 00, and the sample 7,500 mg / 100 ml. The measurement curves drawn on the recording instrument are shown in FIG. 5 · The values obtained are shown in fig. 6 as an adjustment curve and shows that the height of the peaks above the zero line of the measuring instrument is directly proportional to the glucose content of the sample.

Example 2 13 146399

Determination of uric acid by urlease after the equation:

Uric acid + 21 ^ 0 + O2 UI> --— se> allantoin + 1 ^ 2 + CO2

The procedure of Example 1 was used using the same apparatus to determine uric acid. Column S was loaded with uricase on carrier. Column S had a diameter of 1 cm and a filling height of 5 cm. The specific activity of the carrier-bound uricase was 3 U / g.

The apparatus described in Example 1 was used. The inflow velocity to the electrode was 35 cm / sec., The diameter of the input bore in the flow cell shown in FIG. 2, was 1 mm.

In principle, the applicator used had the one shown in FIG. h. The bore 18 in the core 11 had a diameter of 2.5 mm and a length of 13 mm. The applicator consisted of glass.

The individual appliance parts were connected to silicone hoses with a diameter of 1 mm. As buffer, 0.2 M borate buffer of pH 6.5 was used in which 600 U / ml catalase was dissolved.

In the applicator, with a distance of approx. 6 minutes administered samples of uric acid solutions with a specific content. Sample 1 contained 5 mg of uric acid / 100 ml, sample 2 10 mg, sample 3 15 mg, sample k 30 mg, sample 5 ^ 5 mg and sample 6 60 mg / 100 ml.

The measurement curve drawn on the recording instrument is shown in FIG. 7 · The values obtained are shown in fig. 8 as an adjustment curve. It is seen that the height of the peaks above the zero line of the meter is directly proportional to the uric acid content of the sample.

The above example shows that it is also advantageous to carry out the invention with carrier-bound enzymes of inferior specific activity.

Claims (4)

14 146399 A method for quantitatively determining substances in oxygen-containing aqueous solution by oxidation or reduction in the presence of an oxygen-transferring carrier-bound enzyme, in which an aqueous solution of the substance to be determined is introduced into a constant-flow liquid stream of a buffer solution of specific oxygen concentration, after which the solution is passed over the carrier-bound enzyme and the change in oxygen concentration is measured by an oxygen-sensitive electrode, characterized by administering a certain amount of the solution containing the substance to be determined air-free in a constant flow liquid stream of a buffer solution of a certain oxygen concentration in a blocky manner (i.e. as a continuous liquid strand replacing a corresponding liquid strand in the buffer flow) without changing the flow rate, it passes over a carrier bound enzyme and subsequently leads it to the electrode. Process according to claim 1, characterized in that the buffer solution is divided into two partial streams and the partial streams alternately interrupted, and a certain amount of the solution of the substance to be determined is introduced into one partial stream while simultaneously interrupting the second partial stream. Apparatus for carrying out the method according to claims 1 and 2, characterized by a buffer reservoir (R), a sample applicator (A) having at least two at a blocking slider (11) with at least two corresponding bores (17, 18 and 19). ) alternating lockable passages (13a, 15a and 12a, 14a and 20, 16a), one of which bore (18) communicates with an inlet line for the dissolution of the substance to be determined when the corresponding passageway is blocked, a pump (P) with constant performance in the line in front of or behind the applicator (A)., A column (5) with carrier-bound enzyme, and a detector with an oxygen electrode (D) connected to an amplifier (V) and a recording instrument (G), which elements are interconnected in this order by pipelines. Apparatus according to claim 3, characterized in that