CS241022B2 - Molecularly selective sensor and method of its production - Google Patents

Abstract

The invention relates to a molecularly selective sensor suitable for the selective determination of the concentration of compounds (e.g. glucose) dissolved in liquid samples (e.g. blood), as well as a method of its production. The essence of the invention lies in the fact that the opening of the sensor body, which is located on the side of the indicator electrode, is closed by at least one continuous or perforated membrane of natural origin, containing in the surface layer at least one type of enzyme chemically bound in an anchored form, said membrane being in contact with a membrane permeable to gases or to gases and dissolved substances, and an insulating layer is formed between the sensor body and the indicator electrode, while a filter permeable to an electrolyte solution is built into the wall of the cover body. The essence of the method of producing the sensor lies in the fact that at least one type of enzyme is anchored in the upper layer of the membrane of natural origin by means of a chemical bond, after which the membrane containing the anchored enzyme together with the membrane permeable to gases or gases and dissolved; The charged substance is fixed to the opening of a hollow body, into which an electrolyte solution is placed, as well as at least one indicator electrode and at least one reference electrode.

Description

The invention relates to a molecularly selective electroanalytical sensor suitable for the selective determination of the concentration of compounds (e.g. glucose) dissolved in liquid samples (e.g. blood), as well as a method of its production.

Molecular selective electroanalytical sensors are known, suitable for determining compounds dissolved in liquid samples.

The electrochemical properties and possible uses of molecularly selective sensors are known from the literature. As is known, molecularly selective sensors, which can be considered as electrical measuring cells, have some common properties, such as the fact that the sensor carrier, which contains an ion-selective or metal indicator electrode and a reference electrode, is placed in a tubular body filled with an electrolyte solution, one end of which is closed by a membrane permeable to gases and dissolved substances (for example, a dialysis membrane), this membrane being provided with a layer of a gel selective for the substance (for example, an acrylamide gel layer), containing a specifically effective catalyst, i.e. a chemically or physiologically bound enzyme, and is in contact with or layered on a membrane permeable to gases or gases and dissolved substances (for example, a cellophane membrane) and with a layer of a solution containing the selective enzyme in suspended form, as well as a membrane permeable to gases and dissolved substances (for example, a cellophane membrane). The mechanism of action of known molecularly selective sensors is as follows. Molecules of the determined component (molecules of the so-called substrate, for example glucose molecules) and in a given case, molecules of the reagent (oxygen molecules) diffuse into a layer containing a selective enzyme (for example glucose oxidase), serving as a specifically effective catalyst, into the so-called reactive layer, where a chemical reaction occurs. The output signal of the indicator electrode is a unique function of the concentration or reactivity of the reagent participating in the enzymatic reaction, or of one of the participating or emerging components. The local value of the reactivity or concentration depends, if all factors are considered constants, on the reaction rate, which is a unique function of the substrate concentration. Therefore, in a stationary state, it is possible to determine the sample concentration directly by measuring the current strength or emf (electromotive force).

The production of known molecularly selective sensors is a complex, time-consuming and extraordinary knowledge-requiring task, the result of which is reproducible only by chance.

A major disadvantage of these sensors is their short lifespan (e.g., a few days; 1 to 2 weeks), which is related to the unfavorable properties of the reactive layer. Another disadvantage that occurs during use is that the sensors, or rather the gel layers containing the enzyme, must be stored at low temperatures (e.g., between 1 and 2°C) when not in use to maintain enzymatic activity.

Known electrodes are susceptible to damage, have low mechanical strength and a long response time (for example, between 2 and 8 minutes).

Another disadvantage that occurs when using such sensors is that in the amperometric indicator electrode (e.g. platinum) dissolved gas (e.g. oxygen) or an oxide layer formed on the surface, or metal contamination resulting from its use, formed on the surface of the indicator electrode (e.g. silver contamination), or other insulating layers, have an adverse effect on the signal-to-noise ratio, because the active surface of the indicator electrode and thus the signal transmitted by it are reduced.

The above disadvantages are sufficient to prevent sensors providing simple selective measurement techniques from becoming popular in practice and from being available on the market.

The task of this invention is to solve the construction of such a molecularly selective sensor, which would be manufacturable without special demands on professional knowledge, in a short time and reproducibly with a much longer lifetime (for example, half a year to 1 year) than previously known types, and which would exhibit greater mechanical strength and a shorter response time (for example, 20 to 30 seconds).

The essence of the molecularly selective sensor according to the invention lies in the fact that the opening of the sensor body, which is located on the side of the indicator electrode, is closed by at least one continuous or perforated membrane of natural origin, containing in the surface layer at least one type of enzyme chemically bound in an anchored form, said membrane being in contact with a membrane permeable to gases or to gases and dissolved substances, and an insulating layer is formed between the sensor body and the indicator electrode, while a filter permeable to the electrolyte solution is built ^into the wall of the cover body.

The essence of the method for producing a molecularly selective sensor according to this invention consists in anchoring at least one type of enzyme in the upper layer of a membrane of natural origin by means of a chemical bond, after which the membrane containing the enzyme is attached, together with a membrane permeable to gases or gases and dissolved substances, to the opening of a hollow body, into which an electrolyte solution is placed, as well as at least one indicator electrode and at least one reference electrode.

The advantage of the molecularly selective sensor according to the invention lies in the fact that the mechanical strength of albuminous natural membranes (such as the air bladder of fish, the skin of mammals and reptiles, or the cornea and intestinal walls of some animal species) is, despite its thinness of 10 to 15 μτη, advantageous in terms of response time, and they are also advantageous in terms of their structure. In addition, the concentration of active amino groups on membranes formed from amino acids is also optimal and constant in terms of chemical binding of enzymes with an albuminous structure. Thus, it is possible to produce sensors operating in a wide range of concentrations with a short response time. Depending on the anchoring procedure, it is possible to store activated natural membranes in a dry state, or at room temperature, for a very long time (half a year to one year), without the enzyme activity changing noticeably, because it is surrounded by a natural environment.

The permeability of the natural membrane to ions, water molecules and gases is ideal from the point of view of measurement technology. Another advantageous property of natural membranes is the fact that they are permeable to ions or gas molecules, which can occur either as a reagent or as a reaction product, and impermeable to compounds of higher molecular weight, which could possibly affect the measurement result (for example, the substrate itself or other substances contained in the sample), which makes it possible to partially or completely eliminate the so-called hysteresis effect, which often occurs when alternating samples of different concentrations.

The sensor according to the invention can also be arranged in such a way that a membrane with two reactive layers or a plurality of membranes with one or two reactive layers stacked on top of each other is used to make the reactive layer. In this arrangement, a different enzyme is bound in anchored form on each surface of the membrane. In the case of a beta-glucoside electrode, the membrane surface on the sample solution side contains beta-glucosidase, and on the indicator electrode side contains glucose oxidase. The enzyme bound to the first membrane, i.e. beta-glucosidase, enables a hydrolysis reaction between the component to be determined, beta-glucoside (for example, amygdalin or salicin), and water, which also produces glucose, among other things. The enzyme bound to the second membrane, glucose oxidase, catalyzes the reaction between glucose and oxygen. On the indicator electrode located behind the membrane, it is possible to measure a signal proportional to the oxygen concentration. The signal proportional to the decrease in the oxygen concentration is a clear function of the concentration detected in the sample. In this embodiment, on the one hand, it is possible to achieve simplification in terms of the measurement technique and increase selectivity, on the other hand, it is possible to determine even those compounds that do not provide any measurable component in a one-step reaction or do not cause any change in the concentration or reactivity of the measurable components.

The sensor according to the present invention can also be created in such a way that on separate parts of the membrane surface of the natural structure, serving as a reactive zone, or on given parts of the surface, different types of enzymes are bound in a statistically homogeneous distribution, and one part of the surface corresponds to one or more indicator electrodes. With this arrangement, multifunctional molecularly selective sensors can be made without increasing their dimensions, which makes it possible to determine in parallel several components in the same sample solution, or to determine in parallel the concentration of interfering components, the influence of which can be manually or automatically eliminated.

In other embodiments of molecularly selective sensors made according to the invention, for example industrial sensors, the membrane of natural structure is in connection with an inert polymer or fabric network for a greater degree of protection and further increase in the mechanical strength of the reactive zone. In other embodiments, in order to achieve a very short response, the reactive zone containing the enzyme in anchored form is provided in the upper layer of one side of the membrane of natural structure, and in the upper layer of the other side of the membrane of natural structure, a gas-permeable layer or a layer with ion exchange properties is provided according to the impregnation process by crosslinking.

Molecularly selective sensors are made according to the invention in such a way that the surface of a membrane of natural structure is treated with a solution containing an enzyme and a bifunctional reagent containing groups suitable for chemical bonding, for example glutaraldehydes, at ambient temperature. After the bonding reaction is completed, the membrane is placed in a water bath with distilled water, where the components that have retained their solubility in water are separated. After rinsing, the membranes are ready for measurement. Then, in a given case, a metal indicator electrode is prepared. In order to clean the surface of the indicator electrode, it is immersed for several minutes in nitric acid, then rinsed and then placed in a bath containing a reducing agent (for example ascorbic acid), where oxides and oxygen physically dissolved in the metal electrode are removed. The finished membrane, together with a membrane permeable to gases or gases and dissolved substances, is placed on the sensor body, and then both the indicating and reference electrodes are immersed in the electrolyte solution contained in the sensor body in such a way that the surface of the indicating electrode is in the immediate vicinity of the membrane. The electrodes are fixed and the sensor is thus in a state capable of performing measurements.

Example

Fig. 1 shows a partial cross-section of an exemplary embodiment of a glucose-selective sensor according to the present invention. In the cavity of the sensor body 1 there is a buffer solution containing chloride anions 2, into which a platinum indicator electrode 4 and a silver-silver chloride reference electrode 5, mounted in a housing 3, are immersed.

The edge of the sensor body 1 is closed by a gas-permeable polypropylene membrane 6, which contacts the indicator electrode 4. The membrane 6 is in contact with a commercially available pig intestine membrane 7, on which glucose oxidase (E.C.1.1.3.4.) is anchored by means of glutaraldehyde. The membrane 6 and the natural membrane 7 are attached to the body 1 by means of a rubber ring 8. The other end of the sensor body 1 is closed by a plastic cap 9. The indicator electrode 4 and the reference electrode 5 are connected by means of a cable 10 to a polarizing voltage source and the inputs of the ammeter.

The glucose-selective electrode is manufactured as follows. A portion of commercially available salted pig intestine is soaked for 10 to 15 minutes in distilled water. The swollen membrane is attached together with a 15-μm-thick membrane (e.g., a polypropylene membrane) that is gas-permeable, in a stretched state to the edge of the sensor body. The membrane is dried for 20 minutes. Then 60 mg of glucose oxidase (E.C. 1.1.3.4.) is suspended in 0.5 ml of a buffer solution at pH 7.4 and 25 microliters of a 25% glutaraldehyde solution are added. 20 microliters of this homogeneous suspension are spread on the surface of the membrane in an even layer. The membrane is then allowed to dry for 20 minutes, and then the unbound enzyme and the remaining glutaraldehyde are removed by washing with distilled water. The membrane prepared in this way, containing glucose oxidase in the bound form, is in a state capable of being stored or used for measurement.

After the activated membrane is made, a platinum indicator electrode is prepared, inserted into a support in a known manner. The surface of the indicator electrode is soaked for 2 to 3 minutes in a nitric acid solution with a concentration of 6 mol/diji ³ , dropped onto a glass plate. The nitric acid is washed off with distilled water. After washing, the surface of the indicator electrode is soaked in a freshly prepared 2% ascorbic acid solution for 20 to 30 minutes. After soaking, the ascorbic acid is removed from the surface of the indicator electrode by washing with distilled water. The indicator electrode is then ready for measurement. (Wetting in nitric and ascorbic acid must be repeated every 2 to 3 weeks during the measurement.)

After the preparatory operations, the glucose-selective sensor is assembled according to Fig. 1 and then connected to a polarizing voltage source and a current strength measurement unit.

The determination of glucose concentration in a sample can be performed by constructing a calibration curve or by the addition technique. A preferred variant of the addition technique, the so-called multiple sample addition method, is presented below.

It was found that the following relationship exists between the sensing of the measured current strength (Aj) and the glucose concentration of the solution (c):

Δ, = _k - ₍ ij where

V is the maximum reaction rate,

K is the Michaelis constant of the en- reaction.

zym-substrate and k denotes the ratio factor.

From the measured current strength in two solutions containing glucose of known concentration, it is possible to determine the values of V and K, depending on the given circumstances and the sensor, on the basis of which it is also possible to determine the concentration of the nth sample solution from the decreasing current strength, as shown below

C, =. i„k

JC,-, + K) ²

K + V (2).

Based on equation (2), the sample concentration is where a, is the volume of the nth sample and

V _A is the volume of the sample and standard together.

In the case of blood and urine, the measurement is performed using the multiple sample addition method as described below.

6.6 ml of buffer solution with pH 7.4 is measured into a temperature-controlled cell at 37 ⁰ Celsius. The sensor is immersed in the solution. From the measured value of the current strength, after adding 60 to 200 microliters of solutions containing glucose with a concentration of 2 x 10~ ² mol/dm ^{3 ,} the values of V and K are determined numerically or automatically. Then, known volumes of samples are measured into the buffer solution and the decrease in current strength is determined. (The sample volume varies depending on the expected glucose content of the blood sample being tested. For samples with a concentration between 60 and 200 mg/100 ml, the sample volume should be 100 microliters, for a more diluted sample 200 microliters, and for a concentrated sample 50 microliters. For a urine sample with a concentration of 0 to 1,000 mg/100 ml, the measured volume is 25 microliters; for more concentrated samples, a tenfold dilution is performed and 25 to 50 microliters are measured from the diluted sample.) From the measured value of the current strength decrease, the desired concentration is calculated — using equation 2 — numerically or using a computer.

.The values measured by the glucose-selective sensor are given for some samples in Table I,

It is evident from Table I that the variance of the values measured by the glucose-selective sensor is significantly smaller than the variance of known methods, where it often exceeds 10%.

TABLE I

Concentration Number standard parallel solution mg/100 ml measurement 180 11 120 11 90 11

Mean value Variance of measured data in °/o mg/100 ml

180 ' 5

121 3

3

Other molecularly selective sensors can also be made according to this example. Some other examples are given in Table II. The first column shows the enzyme anchored to the natural membrane and connected to the gas permeable membrane. The third column shows the type of indicator electrode.

Determined component Uric acid.

Cholesterol

L-amino acid

D-amino acid

D-galactose

Sulfite

Oxalate

Xanthine o-diphenol p-diphenol hydrogen peroxide

Lactates

Pyruvates

Alcohol

L-Phenylalanine

2-oxoacid

Oxaloacetate

Acetoacetate

L-valine

L-glutamate

L-ornithine

L-lysine

Penicillin

TABLE II Anchored enzyme

Uricase

E.C.1.7.3.3.

Cholesterinoxidase E.C.l.1.3.6.

L-amino acid oxidase E.C.1.4.3.2.

D-amino acid oxidase E.C.1.4.3.3. Galactose oxidase E.C.1.1.3.5.

Sulfite oxidase

E.C.1.8.3.1.

Oxalate oxidase

E.C.1.2.3.4.

Xanthine oxidase

E.C.1.2.3.2.

o-diphenoloxidase

E.C.1.10.3.1.

Catalase

E.C.1.11.1.6.

Lactase oxidase

E.C.1.1.3.2.

Pyruvate oxidase

E.C.1.2.3.3.

Alcohol oxidase

E.C.1.1.3.1.3.

L-phenylalanine

E.C.1.14.3.1.

Pyruvate deoxidase

E.C.4.1.1.1.

Oxaloacetate decarboxylase

E.C.4.1.1.3.

Acetoacetate decarboxylase

E.C.4.1.1.4.

Valine decarboxylase

E.C.4.1.1.14.

Glutamate decarboxylase

E.C.4.1.1.15.

Ornithine decarboxylase

E.C.4.1.1.17.

Lysine decarboxylase

E.C.4.1.1.18.

— Lactamase I. E.C.3.5.2.6.

Indicator electrode Platinum

Platinum

Platinum

Platinum

Platinum

Platinum

Platinum

Platinum

Platinum

Platinum

Platinum

Platinum

Platinum

Platinum

Hydrogen cation-selective electrode Hydrogen cation-selective electrode Hydrogen cation-selective electrode Hydrogen cation-selective electrode Hydrogen cation-selective electrode Hydrogen cation-selective electrode Hydrogen cation-selective electrode Hydrogen cation-selective electrode

Example 2

Fig. 2 shows an exemplary embodiment of a urea-selective sensor in partial cross-section according to the present invention. In the sensor body 1 there is an electrolyte solution 2 containing chloride anions, into which an indicator electrode 4, selective for ammonium cations, and a silver-silver chloride reference electrode 5 are immersed. The indicator electrode 4 is fixed at the end of the sensor body 1 by a cap, impermeable to the electrolyte solution, with a disc-shaped sealing layer 11. The indicator electrode 4 is in contact with a membrane 7 of natural structure, formed by a part of the wall of the air bladder of a fish, which contains urease (E.C.3.5.1.5. J, anchored with glutaraldehyde. The membrane of natural origin 7 is attached to the cover 1 by a rubber ring 8. A filter layer 12 (for example, a ceramic rod), permeable to the electrolyte solution, is placed in the wall 2 of the cover 1. The other end of the cover 1 is closed by a closing flap 9 made of plastic. The indicator electrode 4 and the reference electrode 5 are connected by cable 10 to the inputs of an electronic voltage meter (voltmeter).

The arrangement of the reactive layer of the sensor, selective for urea, on the membrane of natural origin is carried out in the same way as in Example 1. The only difference is that in this case urease (E.C.3.5.1:5.) is anchored on the surface of the membrane. The sensor assembly is carried out as shown in Fig. 2. The concentration of the sample under investigation can be determined in a known manner, either by means of an ems-concentration calibration curve or by means of the addition technique.

Other molecularly selective sensors can also be made according to Example 2. Several other examples are given in Table III. The first column of the table lists the type of molecule to which the sensor selectively responds, the second column lists the anchored enzyme, and the third column lists the selective indicator electrode that must be used for this purpose.

TABLE III

Specified component

Anchored enzyme

Indication electrode

L-dioxyphenylalanine

L-phenylalanine

D-amino acid

D-serine

L-homoserine

L-threomine

L-histidine

Nitrite

L-aminooxidase

E.C.1.4.3.2.

L-aminooxidase

E.C.1.4.3.2.

L-amino acid oxidase E.C.1.4.3.2. D-s'erine dehydratase E.C.4.2.1.14.

Homoserine dehydratase

E.C.4.2.1.15.

Threomine dehydratase

E.C.4.2.1.16.

Histidase

E.C.4.3.1.3.

Nitrite reductase

E.C.1.6.6.4.

Electrode selective for ammonium cations Electrode selective for ammonium cations Electrode selective for ammonium cations Electrode selective for ammonium cations Electrode selective for ammonium cations Electrode selective for ammonium cations Electrode selective for ammonium cations

Example 3

The arrangement of the arginine-selective sensor according to the present invention is, according to the exemplary embodiment, essentially the same as the arrangement of the urea-selective sensor described in Example 2. The arrangement of the sensor presented in Example 3 differs in that at the end of the cover body 1 there are two superimposed membranes 7 of natural origin, which are fixed there by a rubber ring 8. Urease is applied to the membrane placed on the side of the indicator electrode, bound in anchored form, while arginase is bound in anchored form on the other, perforated membrane. The arginine-selective sensor is therefore equipped with two reactive layers.

The production of the arginine-selective sensor is carried out according to the present invention as follows. First, a urea-selective sensor is produced as described in Example 2. Then, on the natural membrane containing urease, another perforated membrane from sheep intestine is placed, on the surface of which a reactive layer containing argininase (E.C.3.5.3.1.) is formed. The production of the reactive layer is carried out as described in Example 1 with the difference that 10 mg of albumin is additionally suspended in the buffer solution.

The arginine-selective sensor can also be manufactured in such a way that only one membrane 7 of natural origin is needed for the sensor arrangement, on the surface of which two types of enzymes, arginase and urease, are anchored in a statistically homogeneous distribution. In this embodiment, the further arrangement and manufacture of the sensor are the same as in the previous case.

The sensor function is based on a two-stage reaction:

L-Arginine + H2O Urea + H2O

Arginase E.C.3.5.3.1. Urease E.C.3.5.1.5.

The arginine concentration of the sample under investigation can be determined in a known manner, namely by means of a calibration curve or by using the addition technique.

Other molecularly selective sensors that can be produced in a similar manner as described in Example 3, i.e. with two reactive layers or with one reactive layer containing two types of enzymes distributed statistically homogeneously, are given in Table IV.

L-Ornithine + Urea Acid. carbon + ammonia CO2 NHs

The first column lists the molecule detectable by the sensor, the second column lists the enzymatic catalyst anchored on the outer membrane of natural origin. The third column lists the enzymatic catalysts located closer to the indicator electrode, i.e. on the inner membrane. The fourth column lists the ranges of the indicator electrodes.

TABLE IV £5_tanpýpvaiiá . Type of enzyme anchored on the membrane surface component. \ first second Indication electrode.

Iňbsin -Uridine nucleosidase Xanthine oxidase Platinum

Adenosine E.C.3.2.2.2. Adenosine nucleosidase E.C.3.2.2.a. Guanosine Guanosine phosphorylase E.C.2.4.2.15. Guanin' Krestinin ' Guanase E.C.3.5.4.3. Christininase E.C.3.5.3.3. beta-D-glucosides, disaccharides, amygdalin, salicin alpha-D-glucosides (e.g. maltose) D-glucose-6-phosphates D-glucose-6-phosphates Barbiturates ' beta-glucosidase E.C.3.2.1.21. alpha-glucosidase E.C.3.2.1.20. Glucose-l-phosphatase E.C.3.1.3.10. / E.C.3.1.3.9. Glucose-6-phosphatase Barbiturase E.C.3.5.2.1.

Example 4 In Example 4, an exemplary embodiment and fabrication of bifunctional molecularly selective sensors fabricated according to the present invention is described.

- In the cover body 1 there is a buffer solution 2 containing chloride anions, into which two platinum indicator electrodes are immersed, which are individually built in each case in one carrier 3. In contact with the surfaces of the indicator electrodes 4 is a polypropylene membrane 6 permeable to gases, fixed to the inside of the cover body 1. The surface of the membrane 6 is covered with a piece of leather, a membrane of natural origin. On the part of the surface of the membrane 7 of ^natural origin, which is in contact with one of the indicator electrodes 4,

E.C.1.2.3.2. Adenine deaminase Selective electrode E.C.3.5.4.2. to ammonium or hydrogen cations Guanáza Selective electrode E.C.3.5.4.3. to ammonium or hydrogen cations _ Xanthine oxidase E.C.1.2.3.2. Platinum Urease Selective electrode E.C.3.5.1.5. to ammonium or hydrogen cations Glucose oxidase E.C.l.1.3.4. Platinum Glucose oxidase E.C.1.1.3.4. Platinum Glucose oxidase E.C.1.1.3.4. Platinum ^; Glucose oxidase E.C.1.1.3.4. Platinum Urease Selective electrode E.C.3.5.1.5. to ammonium or hydrogen cations

or on the same part of the membrane surface on the opposite side is bound by anchoring D-amino acid oxidase and on the other part of the membrane surface which is in contact with the second indicator electrode, or on the same part of the membrane surface on the opposite side is bound by anchoring L-amino acid oxidase.

Other details of the bifunctional molecularly selective sensor arrangement are the same as the sensor arrangement shown in Example 1.

The production of reactive layers containing amino acid oxidase is carried out as follows. A wet skin membrane is stretched onto a gas-permeable polypropylene membrane. It is then allowed to dry for 30 minutes at a temperature of 241022

Then, 10 microliters of a phosphate buffer solution with a pH of 8.3, each 0.1 ml of which contains 5 mg of D-amino acid oxidase (E.C.1.4.3:3 Sygma, crystalline) in suspended form, are distributed over a portion of its surface, and then 10 microliters of a 25% glutaraldehyde solution are added. Then, 10 microliters of a phosphate buffer solution with a pH of 6.5, each 0.1 ml of which contains 10 mg of L-amino acid oxidase (E.C.1.4.3.2. Sygma IV] in suspended form, are distributed over another portion of the membrane surface, and then 10 microliters of a 25% glutaraldehyde solution are added.

The further steps of the production process are the same as described in Example 1. Using a bifunctional molecularly selective sensor, parallel selective determination of D and L isomers of amino acids is possible. The measurement is carried out in such a way that the selective sensor is calibrated first with a solution of the L-form and only then with a solution of the D-form of the determined amino acid (for example, phenylalanine). During calibration, the sensor is placed in a stirred phosphate buffer solution (pH 8) in a standard solution stirred at a constant speed with the same pH value, and the change in current intensity (Aj) resulting from the effect of a -0.6 V polarization voltage is measured. If we interpret the values of Aj as a function of the concentration of the standard solutions, it is possible to make calibration curves and with their help, after measuring A s, determine the ratio _of optical isomers of different amino acids.

The use of a complex sensor made according to the above example is extremely advantageous in investigating the efficiency of resolution processes.

The addition techniques described in Example 1 can also be used to determine the ratio of isomers. Further, advantageously usable bifunctional molecularly selective sensors according to the present invention can be prepared according to Example 4. This is summarized in Table V.

The second column of the table lists the names of enzymes anchored on one part of the surface, and the fourth column lists the names of enzymes anchored on the other part of the surface.

TABLE V

Specified component Enzyme anchored on one side of the membrane Glucose glucose oxidase E.C.1.1.3.4. Glucose glucose oxidase E.C.1.1.3.4. Urea urease E.C.3.5.1.5. Glucose glucose oxidase E.C.1.1.3.4. Glucose glucose oxidase E.C.1.1.3.4. Urea urease E.C.3.5.1.5. Glucose glucose oxidase E.C.1.1.3.4.

Determined Enzyme anchored to the component of the second part of the membrane

Example 5

Example 5 describes an exemplary arrangement and production of a multifunctional molecularly selective sensor according to the present invention, suitable for parallel determination of various amino acids.

The arrangement of the multifunctional sensor is similar to the bifunctional sensor described in Example 1. The difference lies in the fact that it does not contain a membrane 6 permeable to gases, and in addition, in the electrolyte solution 2, located in the cover body 1, three glass electrodes selective for hydrogen cations are immersed, which are in contact with a membrane 7 of natural origin, on which three different reactive layers are made on three well-separated parts of the surface, in which

alcohol alcohol oxidase E.C.1.1.3.1.9. mannose hexose oxidase E.C.1.1.3.5. creatine creatininase E.C.3.5.3.3. uric acid uricase E.C.1.7.3.3. L-phenylalanine L-phenylalaninease E.C.1.14.3.1. Arginine Arginine decarboxylase E.No.4.1.1.19. Amygdalin Glucose oxidase E.C.3.2.1.21. glucose oxidase E.C.1.1.3.4. are bound in anchored form three selecti

amino decarboxylases (L-lysine decarboxylase, L-tyrosine decarboxylase, L-phenylalanine decarboxylase). The reactive layers are prepared in accordance with the description in Example 1.

The molecularly selective sensor, made in the exemplary embodiment, can be used to selectively determine the contents of L-tyrosine, L-lysine and phenylalanine in samples containing amino acids. The determination can be performed using three calibration curves of potential and concentration corresponding to the three amino acids.

It is possible to make other similar variants according to the multifunctional molecularly selective sensor described as an example. Individual parts of the reactive layer contain the following enzymes in anchored form: L-arginine decarboxylase, L-glutamine decarboxylase, L-glutamic acid decarboxylase, L-histidine decarboxylase, urease.

Claims (5)

Subject 1. Molecular selective sensor for determining the concentration of molecules or ions in solutions, consisting of a capsule filled with an electrolyte solution carrying a potentiometric, voltamperic indicator electrode and a reference electrode immersed in an electrolyte solution, gas permeable membranes or gas and solutes enclosing an aperture located on the side of the indicating electrode, wherein the membrane is in contact with a gel layer comprising a chemically bound enzyme selective for the substance of interest and the electrodes are connected to a voltage source and current meter or a voltage measuring unit, characterized in that the opening of the sensor body (1) located on the side of the indicating electrode (4) is closed by at least one continuous or perforated membrane (7) of natural origin, containing at least one type of enzyme bound chemically in anchored form in the surface layer; (7) is in contact with the membrane (6) permeable to gases or gases and solutes, and an insulating layer (11) is formed between the sensor body (1) and the indicating electrode (4), while in the wall of the cover body (1) a filter permeable for the electrolyte solution is built in. VYNALEZU Molecular selective sensor according to claim 1, characterized in that the membrane (7) of natural origin is in contact with a protective net or fabric made of an inert substance. Molecular selective sensor according to Claims 1 and 2, characterized in that it comprises a plurality of indicator electrodes (4). Molecular selective sensor according to Claims 1 to 3, characterized in that a gas-permeable or gas-permeable layer and a solute are formed in the upper layer of the membrane (7) which contains no enzyme in anchored form. 5. A method for producing a molecular selective sensor according to any one of claims 1 to 4, wherein at least one type of enzyme is anchored in the upper layer of the membrane of natural origin by means of chemical bonding. fastens to the opening of the hollow body into which the electrolyte solution and the at least one indicating electrode and at least one reference electrode are placed.