CA1218289A - Method for performing and tracing enzymatic reactions and a device for carrying out the said method - Google Patents
Method for performing and tracing enzymatic reactions and a device for carrying out the said methodInfo
- Publication number
- CA1218289A CA1218289A CA000459834A CA459834A CA1218289A CA 1218289 A CA1218289 A CA 1218289A CA 000459834 A CA000459834 A CA 000459834A CA 459834 A CA459834 A CA 459834A CA 1218289 A CA1218289 A CA 1218289A
- Authority
- CA
- Canada
- Prior art keywords
- enzymatic
- immobilized
- reactor
- concentration
- stirrer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 238000000034 method Methods 0.000 title claims description 34
- 238000006911 enzymatic reaction Methods 0.000 title claims description 25
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 29
- 239000000463 material Substances 0.000 claims abstract description 24
- 239000000126 substance Substances 0.000 claims abstract description 19
- 108010093096 Immobilized Enzymes Proteins 0.000 claims abstract description 10
- 238000005259 measurement Methods 0.000 claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims description 40
- 108090000790 Enzymes Proteins 0.000 claims description 40
- 239000011541 reaction mixture Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 3
- 235000019422 polyvinyl alcohol Nutrition 0.000 claims description 3
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 2
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims description 2
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims description 2
- 230000008901 benefit Effects 0.000 abstract description 6
- 239000011888 foil Substances 0.000 abstract description 4
- 239000004744 fabric Substances 0.000 abstract description 2
- 239000010410 layer Substances 0.000 abstract 2
- 229940088598 enzyme Drugs 0.000 description 39
- 239000000523 sample Substances 0.000 description 20
- 239000000758 substrate Substances 0.000 description 13
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 239000001301 oxygen Substances 0.000 description 12
- 229910052760 oxygen Inorganic materials 0.000 description 12
- 230000008859 change Effects 0.000 description 11
- 108010015776 Glucose oxidase Proteins 0.000 description 9
- 239000004366 Glucose oxidase Substances 0.000 description 9
- 229940116332 glucose oxidase Drugs 0.000 description 9
- 235000019420 glucose oxidase Nutrition 0.000 description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 239000008103 glucose Substances 0.000 description 8
- 238000009792 diffusion process Methods 0.000 description 7
- 230000007423 decrease Effects 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 238000011088 calibration curve Methods 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 102000016938 Catalase Human genes 0.000 description 3
- 108010053835 Catalase Proteins 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 239000004372 Polyvinyl alcohol Substances 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 2
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 2
- 102000016679 alpha-Glucosidases Human genes 0.000 description 2
- 108010028144 alpha-Glucosidases Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 229940068984 polyvinyl alcohol Drugs 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 238000005496 tempering Methods 0.000 description 2
- 229940116269 uric acid Drugs 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- 108010073450 Lactate 2-monooxygenase Proteins 0.000 description 1
- 108010092464 Urate Oxidase Proteins 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- MGNCLNQXLYJVJD-UHFFFAOYSA-N cyanuric chloride Chemical compound ClC1=NC(Cl)=NC(Cl)=N1 MGNCLNQXLYJVJD-UHFFFAOYSA-N 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 239000001573 invertase Substances 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000002311 subsequent effect Effects 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 229940005267 urate oxidase Drugs 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Clinical Laboratory Science (AREA)
- Molecular Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE:
An enzymatic material is immobilized onto the surface of a removable stirrer, e.g. magnetic or mechanical, or alternatively on a surface of another member which is removably fixed in a reactor. The enzymatic material may either be immobilized directly or indirectly onto the strir-rer or the other removable member. The enzymatic material is used in a quantity sufficient such that the concentration of the determined substance on the surface of the enzymatic layer may be maintained at a value of 0 to 3% during the course of a plurality of measurements. The enzymatic mate-rial may be fixed onto the surface of the stirrer of other removable member either directly or by means of a suitable layer, foil or cloth. The fixing of enzymatic material onto the stirrer or other removable member obviates all drawbacks of enzymatic electrodes, while providing the advantage of numerous applications of the immobilized enzyme, as well as specific determinations.
An enzymatic material is immobilized onto the surface of a removable stirrer, e.g. magnetic or mechanical, or alternatively on a surface of another member which is removably fixed in a reactor. The enzymatic material may either be immobilized directly or indirectly onto the strir-rer or the other removable member. The enzymatic material is used in a quantity sufficient such that the concentration of the determined substance on the surface of the enzymatic layer may be maintained at a value of 0 to 3% during the course of a plurality of measurements. The enzymatic mate-rial may be fixed onto the surface of the stirrer of other removable member either directly or by means of a suitable layer, foil or cloth. The fixing of enzymatic material onto the stirrer or other removable member obviates all drawbacks of enzymatic electrodes, while providing the advantage of numerous applications of the immobilized enzyme, as well as specific determinations.
Description
21~3~B~
The invention relates to a method for performing and analysing enzymatic reactions which makes use of immo-bilized enzymes. The invention also relates to a device for carrying out the said method.
The quantitative determination of the concentra-tion of a substrate relies on an experimental determination of a calibrating curve. It is advantageous for the cali-bration to be linear and to be stable during the course of time. Kinetic and stabilized calibration methods are being used. The kinetic methods seek to calibrate a para-meter characterizing a rate of change of a traced ~uantity, which is monitored in a reaction mixture after a sample has been added. For example, an initial value of a de-pendence gradient of the traced quantity ~s time is em-ployed for which the traced quantity achieves, after adding the sample, a value having been determined in ad-vance; alternatively one applies a value of the quantity wnich is achieved over time, determined in advance, after the sample has been added As to stabilized methods, a calibration dependence is determined from a stabilized value of the traced quantity, achieved in the reaction mixture after adding a sample. Kinetic methods of cali-bration are advantageous, since they provide a quick deter-mination of the respective substrate. However, they do have the drawback thai the rate of traced change may depend upon the kinetics of the enzymatic reaction itself, which may be considerably altered as a function of the falling activity of the enzyme.
T~ methods of adding enz~mes into a reaction tank are usually used. According to one method an enzyme or enzymes, dissolved or suspended in a suitable buffer, are added to a medium consisting of a buffer and cofactors. Then there are added: a tested sample, glucose, blood, plasma, etc., and one records, by means of a sensing element, the ~.
~18~l3'3 course of an enzymatic reaction, e.g. a decrease of oxygen in a reaction tank by means of an oxygen electrode. By comparing the results to the calibration curve, one deter-mines the concentration of the substance in the sample.
For thismethod of addition, the enzyme is homogenously dispersed in the medium. Once the reaction is over, the enzyme is removed together with the reaction medium. For any other determination one must add a new amount of the enzyme. The main drawback of this method resides in the high consumption of axpensive enzymes, which are used up after a single application.
As to the other method, an enzyme or enzymes are immobilized on the outer surface of a membrane covering the sensing element. Alternatively, another membrane or cloth is put on the membrane covering *he sensing element, and the enzyme is fixed thereto in a suitable way. In such a way a so called enzyme electrode is created. The immobilized enzyme may be used for many analyses. The main drawback of enzyme electrodes resides in the fact that the enzymatic reaction results in a concentration gradient of the analysed product of the reaction or substrate in the immediate neighbourhood of the electrochemical sensor. Since the signal level of the sensor depends upon the kinetics of transport of the analysed substance through the layer of the fixed enzyme, the sensor participates in the production its signal level. The transport of the analysed component through such a layer, and thus the signal of the probe, is determined by an inner diffusion of substances, taking part in the reaction, in the layer, and by the velocity of the enzymatic reaction, ~hich is also dependent upon the concentration and activity of the fixed enzyme in the layer. Accordingly, calibration properties of enzyme electrodes depend both upon kinematic paràmeters of the enzymatic reaction and upon parameters characterized lZ~8Z~39 by the diffusion transport of individual components in the membranes covering the sensor. These parameters may be affected by the kinds of preparation of the probe with a fixed enzyme, e.g. they may depend upon the quant~ty of the enzyme fixed in a volume unit of a carrier, upon its activi ty, etc. The diffusivity of components through the carrier is also affected not only by changes in the quantity of the fixed enzyme, but by the degree of netting of the carrier. As a probe is used, the activity of the enzyme decreases continuously. The said effects, which consider-ablv affect the signal level of the probe, cannot be easily described in simple equations and that is why they are not suitable for automatically recognizing the signal.
The mentioned facts result in the following drawbacks of the hitherto known analyzers making use of enzymatic electrodes. Usually the difficult of preparing multilayer membranes, makes it impossible to be able to recommence the taking of measurements. The probes usually cannot be prepared for stock and kept under deep temperatures till one needs them. The taking of (useful) measurements is complicated by the dependence of the signal level of the probe upon the kinetics of the-enzymatic reaction and upon the internal diffusion of all reacting components in the layer of the fixed enzyme, resulting from the geometry of the system.
Since the signal of the probe is determined by diffusion processes in layers closely adjacent to the electrochemical sensor~ the signal is very sensible to small changes in the geometry of the said layers. Such changes may be caused by slight deformations of elastic layers, e.g.
by a mechanical contact, by an intensive mixing, by a shock of an electromagnetic mixer onto the sensor, etc. Thus the signal is subject to considerable instability neces-sitating frequent recalibration and a very complicat~d :12~ ,89 mathematical description of the function of the enzymatic electrode. When applying a micro computer for recognising the signal of the probe, a complicated softwear and large memory capacity are needed.
In accordance with the present invention the mentioned drawbacks may be obviated by a method for per-forming and analysing enzymatic reactions, wherein the enzymatic material is immobilized on the surface of a part separate from the sensor.
Thus the present invention provides a method for performing and analysing enzymatic reactions making use of immobilized enzymes characterized in that the enzymatic reaction or reactions are performed in a reactor wherein the enzymatic material is immobilized on a surface in the reactor which is separate from the sensor of a measuring device and in a quantity sufficient to maintain the con-centration of the tested substance on the surface of the enzymatic material during a plurality of measurements at from 0 to 3% with respect to the concentration of the initial solution.
In particular, the present invention provides a method for performing and analysing enzymatic reactions making use of immobilized enzymes, characterized in that the enzymatic reaction or reactions are performed in a reactor wherein the enzymatic material is immobilized on the surface of a removable stirrer and/or on the surface of one or more other removably $ixed members, the stirrers and said members being disposed inside the reactor separately from the sensor of a measuring device and in a quantity sufficient to maintain the concentration of the tested substance on the surface of the enzymatic material during a plurality of measurements at from 0 to 3~, with respect to the concentration of the original solution.
As indicated above, in accordance with the present :~Z18~39 invention e~zymatic reactions are performed in a reactor, enzymatic material is fixed onto the surface of the mixer or on the surface of another unmovable part, which may be easily taken of~, which is installed inside the reactor and separated from the sensor of a measuring device, in a quantity being sufficient for keeping up the concentra-tion of the determined substance on the surface of the enzymatic layer during many measurements at values from 0 and 3~ with respect to the concentration of the initial solution. According to the invention a substance increasing viscosity may be included in the liquid reaction mixture, e.g. carboxymethylcelulose, or polyvinylalcohol or mixtures thereof.
A device for carrying out the method according to the invention may consist of a reactor provided with a mixer, advantageously magnetic one, on the surface of which there is fixed a layer of enzymatic material. In an alternative embodiment, the device may consist of a reactor, inside which there are situated one or more unmovable parts, which may be taken off, and which are provided with a layer of a fixed enzym-atic material.
The main advantage of the method according to the invention resides in its wide application L for the determination of glucose in body liquids (e.g. blood, serum, plasma, urine) by oxydationofglucose with oxygen in the presence of enzymes of glucoseoxidase and catalase by analysing oxygen consumption by means of an oxygen electrode.
The method according to the invention in combination with an oxygen electrode may be applied for a quantitative determination of other substances as well, e.g. of lactic acid, uric acid, l-aminoacids, d-aminoacids, galactose and other substrates in combination with the respective specific oxidase. The method according to the invention may also lZ~ 39 be used for a quantitative determination of substances for which a respective oxidase is not known, (e.g. for deter-mining sacharose, maltose or glycogen~ if several different enzymes are applied, the subsequent effect of which pro-vides an oxidable substrate. To determine sacharose, oneapplies invertase and glucose-oxidase; for determining maltose an enzyme of maltase and glucose-oxidase, for determining glycogene enzymes,amylase, maltase and glucose-oxidase. The method in combination with another kind of sensor, e.g. with a pH electrode, may be applied for a quantitative determination of unoxidable substrates. The method according to the invention may be used not only for analytic purposes, but for production purposes as well, e.g. for purification of solutions of medi~inals or drinks from undesired foreign substances or products, and in general , anywhere, where it is possible by means of an enzyme or enzymes to provide a desired change and by means of a physical parameter to inspect a process or finish of an enzymatic reaction. Such physical parameters may be represented e.g. by an oxygen concentration, concentration of hydrogen peroxide, fluorescence, pH, conductivity, extinction, colour, etc.
The device according to the invention provides the following advantages. The body of the stirrer may be easily taken out, as well as the other removably fixed members provided with immobilized enzyme. Thus they may be easily changed or renewed, as eventually it may be desired that the outer layer of fixed enzymatic material be changed without changing the characteristics of the sensor used for tracing the process of the reaction. The mixer or other object with the fixed enzyme or foil with fixed enzym-atic material for fixing onto the surface of the mixer or removable object may be prepared for stock and kept, without changing their activity, for several months.
8S~
By changing the mixer with enzymatic ~aterial immobilized on its surface for a mixer with another enzym-atic material fixed on its surface, one may analyse another substrate in the same reactor by means of the same sensor.
In a single sam~le, in the same reactor with a single sensor there may be determined, by a change of mixer with the respective immobilized enzymes, several substances, e.g. glucose in blood plasma with immobilized glucose-oxidase, by a change for an object with immobilized lactate-oxidase: lactic acid, and uric acid by a change for an object with immobilized urate-oxidase, all without changing the volume of the reaction tank or sensor.
By changing revolutions of the mixer or by changing the viscosity of the reaction mixture by adding substances affecting viscosity, e.g. of carboxymethylcellulose or poly-vinyl alcohol, one may control th~ effect of the outer diffusion on the rate of change of the traced quantity, and in this way one may change the calibration scope of the analyser. One achieves the ~one of the control function of the external diffusion of the substrate, if the concen-tration of the substrate on the surface of the fixed enzyme is kept at a value going near to zero, in any case a value lower than 3~ of the value of the initial concentration of the substrate in the sample. This zone may be achieved by various processes, e.g. by decressing the mixer revo-lutions,which provides an increased thickness of a liquid film on the surface of the layer with enzymatic material by increasing the sample viscosity of the tested liquid, which also provides a thickening of the liquid film and more over the diffusivity of the substrate decreases; or by increasing the activity of the immobilized enzyme. If one uses the device for controlling the external diffusion of the determined substance, other advantages may come in question:
~Z~8'~9 ~ simple mathematical description of the rate of change of the analysed quantity in dependence upon time, may be represented for a closed reactor by an exponential dependence of the value of the analyzed quantity, e.g. of oxygen concentration, vs time. This character of the dependence is the same, for any enzyme used or any concen-tration of any substance to be analyzed. A micro-computer, may be advantageously utilized for recognizing the signal because softwear equipment is simple and identical for a lot of instruments for determining various substrates.
A calibration dependence, residing in the value of the initial rate of change or the analysed quantity, e.g. rate of change of oxygen concentration, is then linear, but the gradient of the linear dependence is not dependent upon the activity of the enzyme; of course, if the concentration or activity of the enzyme is sufficiently high. The activity of the enzyme determines only the range, in which the calibration dependence is linear. The range of linear-ity decreases in dependence upon the decreasing activity 20 of the enzyme. A long-time stability of the device, viz.
of its calibration, may be achieved by applying a high concentration of the fixed enzyme. In this way any decrease of the activity does not affect the calibration, if for example the enzyme is able to keep up the concen-tration of the determined substrate on the surface of theenzymatic layer in the value near to zero at the upper limit of the range of the device. The increase of vis-cosity of the solution, or the decrease of mixer revolutions may provide an enlarging of the linear zone of calibration.
The calibration dependence is not sensible to mechanical shock. The mixer or the said takable out parts may be taken out of the reactor, taken in hand and put again into the reactor without changing the calibration of the device.
In order that the invention may be clearly under-121~3Z89 stood and readily carried into effect, a preferred embodi-ment thereof is, by way of example, hereinafter more fully described and illustrated in the accompanying drawings, in which: Figure 1 shows a schematic sketch of the device for carrying out the method, and Figure 2 shows a calibration curve.
Example:
A device for performing the method according to the invention is shown in Fig. 1. It consists of a tempering or temperature jacket 1, oxygen probe 2, the sensor of which is separated from the sample in the reactor 3 by means of a membrane 4 permeable to oxygen. Inside the tempering jacket 1, there is situated a magnetic stirrer 5, onto which a foil 7 with immobilized enzymes is fixed by means of a ring 8. The magnetic stirrer 5 is driven by means of a magnet 6. Directly on the polyacryl-amide surface of the magnetic stirrer 5 or on the foil 7 of the same material, as it is shown in Fig. 1, a mixture of glucose-oxidase and catalase in a total quantity of 20 international units is immobilized by means of any known method, e.g. by means of cyanuric chloride or carbodiimide or by combination of an enzyme with a suitable inert protei~
~nd glutaraldehyde. ~ sample - solution of buffer ~ith pH 6,6 comprising glucose, the quantity of which is to be determined, was put into the reactor. In the table there is in the first column the initial gradient of dependence of the signal of the oxygen probe 2 in any units, in the column 2 the initial concentration of glucose in the reaction mixture, in milimvls, and in the column 3 there is the number of micromols of the original solution. The table sho~s evidently that there was- achieved a wide zone of linearity of the calibration curve in the range 0 up to 0,7407 mM of the initial concentration of glucose. The _ g _ :~Z18~39 respective calibration curve is shown in Fig. 2.
Enzymes were fixed on a nylon mesh having size of eyes 50 um of area limited by a ring 8 of dia 10 mm.
Onto this area there were applied approx. 20 international units of crude glucose-oxidase comprising catalase as well.
The preparation of the solution for fixing glucose-oxidase onto the nylon mesh is carried out as follows: 100 ul of the mixture is to be prepared: 5 mg of glucose-oxidase dissolved in 50 ul of water, one adds 50 ul of 10% of beef albumin and 20 ml of 2% glutaraldehyde in 0,5 M of phosphate buffer pH 6,6. On the mesh into the ring 8 of dia 10 mm there is applied 15 up to 20 ul of this mixture and one let it dry up.
:12~ 8'~
T A B L E
Initial gradient Initial concentrat- ul of the ori-of dependence of ion of glucose in ginal solution the oxygen probe the reaction mixture of glucose 0.4 0.0185 1.25 1.0 0.0481 3,25 1.6 0.0741 5
The invention relates to a method for performing and analysing enzymatic reactions which makes use of immo-bilized enzymes. The invention also relates to a device for carrying out the said method.
The quantitative determination of the concentra-tion of a substrate relies on an experimental determination of a calibrating curve. It is advantageous for the cali-bration to be linear and to be stable during the course of time. Kinetic and stabilized calibration methods are being used. The kinetic methods seek to calibrate a para-meter characterizing a rate of change of a traced ~uantity, which is monitored in a reaction mixture after a sample has been added. For example, an initial value of a de-pendence gradient of the traced quantity ~s time is em-ployed for which the traced quantity achieves, after adding the sample, a value having been determined in ad-vance; alternatively one applies a value of the quantity wnich is achieved over time, determined in advance, after the sample has been added As to stabilized methods, a calibration dependence is determined from a stabilized value of the traced quantity, achieved in the reaction mixture after adding a sample. Kinetic methods of cali-bration are advantageous, since they provide a quick deter-mination of the respective substrate. However, they do have the drawback thai the rate of traced change may depend upon the kinetics of the enzymatic reaction itself, which may be considerably altered as a function of the falling activity of the enzyme.
T~ methods of adding enz~mes into a reaction tank are usually used. According to one method an enzyme or enzymes, dissolved or suspended in a suitable buffer, are added to a medium consisting of a buffer and cofactors. Then there are added: a tested sample, glucose, blood, plasma, etc., and one records, by means of a sensing element, the ~.
~18~l3'3 course of an enzymatic reaction, e.g. a decrease of oxygen in a reaction tank by means of an oxygen electrode. By comparing the results to the calibration curve, one deter-mines the concentration of the substance in the sample.
For thismethod of addition, the enzyme is homogenously dispersed in the medium. Once the reaction is over, the enzyme is removed together with the reaction medium. For any other determination one must add a new amount of the enzyme. The main drawback of this method resides in the high consumption of axpensive enzymes, which are used up after a single application.
As to the other method, an enzyme or enzymes are immobilized on the outer surface of a membrane covering the sensing element. Alternatively, another membrane or cloth is put on the membrane covering *he sensing element, and the enzyme is fixed thereto in a suitable way. In such a way a so called enzyme electrode is created. The immobilized enzyme may be used for many analyses. The main drawback of enzyme electrodes resides in the fact that the enzymatic reaction results in a concentration gradient of the analysed product of the reaction or substrate in the immediate neighbourhood of the electrochemical sensor. Since the signal level of the sensor depends upon the kinetics of transport of the analysed substance through the layer of the fixed enzyme, the sensor participates in the production its signal level. The transport of the analysed component through such a layer, and thus the signal of the probe, is determined by an inner diffusion of substances, taking part in the reaction, in the layer, and by the velocity of the enzymatic reaction, ~hich is also dependent upon the concentration and activity of the fixed enzyme in the layer. Accordingly, calibration properties of enzyme electrodes depend both upon kinematic paràmeters of the enzymatic reaction and upon parameters characterized lZ~8Z~39 by the diffusion transport of individual components in the membranes covering the sensor. These parameters may be affected by the kinds of preparation of the probe with a fixed enzyme, e.g. they may depend upon the quant~ty of the enzyme fixed in a volume unit of a carrier, upon its activi ty, etc. The diffusivity of components through the carrier is also affected not only by changes in the quantity of the fixed enzyme, but by the degree of netting of the carrier. As a probe is used, the activity of the enzyme decreases continuously. The said effects, which consider-ablv affect the signal level of the probe, cannot be easily described in simple equations and that is why they are not suitable for automatically recognizing the signal.
The mentioned facts result in the following drawbacks of the hitherto known analyzers making use of enzymatic electrodes. Usually the difficult of preparing multilayer membranes, makes it impossible to be able to recommence the taking of measurements. The probes usually cannot be prepared for stock and kept under deep temperatures till one needs them. The taking of (useful) measurements is complicated by the dependence of the signal level of the probe upon the kinetics of the-enzymatic reaction and upon the internal diffusion of all reacting components in the layer of the fixed enzyme, resulting from the geometry of the system.
Since the signal of the probe is determined by diffusion processes in layers closely adjacent to the electrochemical sensor~ the signal is very sensible to small changes in the geometry of the said layers. Such changes may be caused by slight deformations of elastic layers, e.g.
by a mechanical contact, by an intensive mixing, by a shock of an electromagnetic mixer onto the sensor, etc. Thus the signal is subject to considerable instability neces-sitating frequent recalibration and a very complicat~d :12~ ,89 mathematical description of the function of the enzymatic electrode. When applying a micro computer for recognising the signal of the probe, a complicated softwear and large memory capacity are needed.
In accordance with the present invention the mentioned drawbacks may be obviated by a method for per-forming and analysing enzymatic reactions, wherein the enzymatic material is immobilized on the surface of a part separate from the sensor.
Thus the present invention provides a method for performing and analysing enzymatic reactions making use of immobilized enzymes characterized in that the enzymatic reaction or reactions are performed in a reactor wherein the enzymatic material is immobilized on a surface in the reactor which is separate from the sensor of a measuring device and in a quantity sufficient to maintain the con-centration of the tested substance on the surface of the enzymatic material during a plurality of measurements at from 0 to 3% with respect to the concentration of the initial solution.
In particular, the present invention provides a method for performing and analysing enzymatic reactions making use of immobilized enzymes, characterized in that the enzymatic reaction or reactions are performed in a reactor wherein the enzymatic material is immobilized on the surface of a removable stirrer and/or on the surface of one or more other removably $ixed members, the stirrers and said members being disposed inside the reactor separately from the sensor of a measuring device and in a quantity sufficient to maintain the concentration of the tested substance on the surface of the enzymatic material during a plurality of measurements at from 0 to 3~, with respect to the concentration of the original solution.
As indicated above, in accordance with the present :~Z18~39 invention e~zymatic reactions are performed in a reactor, enzymatic material is fixed onto the surface of the mixer or on the surface of another unmovable part, which may be easily taken of~, which is installed inside the reactor and separated from the sensor of a measuring device, in a quantity being sufficient for keeping up the concentra-tion of the determined substance on the surface of the enzymatic layer during many measurements at values from 0 and 3~ with respect to the concentration of the initial solution. According to the invention a substance increasing viscosity may be included in the liquid reaction mixture, e.g. carboxymethylcelulose, or polyvinylalcohol or mixtures thereof.
A device for carrying out the method according to the invention may consist of a reactor provided with a mixer, advantageously magnetic one, on the surface of which there is fixed a layer of enzymatic material. In an alternative embodiment, the device may consist of a reactor, inside which there are situated one or more unmovable parts, which may be taken off, and which are provided with a layer of a fixed enzym-atic material.
The main advantage of the method according to the invention resides in its wide application L for the determination of glucose in body liquids (e.g. blood, serum, plasma, urine) by oxydationofglucose with oxygen in the presence of enzymes of glucoseoxidase and catalase by analysing oxygen consumption by means of an oxygen electrode.
The method according to the invention in combination with an oxygen electrode may be applied for a quantitative determination of other substances as well, e.g. of lactic acid, uric acid, l-aminoacids, d-aminoacids, galactose and other substrates in combination with the respective specific oxidase. The method according to the invention may also lZ~ 39 be used for a quantitative determination of substances for which a respective oxidase is not known, (e.g. for deter-mining sacharose, maltose or glycogen~ if several different enzymes are applied, the subsequent effect of which pro-vides an oxidable substrate. To determine sacharose, oneapplies invertase and glucose-oxidase; for determining maltose an enzyme of maltase and glucose-oxidase, for determining glycogene enzymes,amylase, maltase and glucose-oxidase. The method in combination with another kind of sensor, e.g. with a pH electrode, may be applied for a quantitative determination of unoxidable substrates. The method according to the invention may be used not only for analytic purposes, but for production purposes as well, e.g. for purification of solutions of medi~inals or drinks from undesired foreign substances or products, and in general , anywhere, where it is possible by means of an enzyme or enzymes to provide a desired change and by means of a physical parameter to inspect a process or finish of an enzymatic reaction. Such physical parameters may be represented e.g. by an oxygen concentration, concentration of hydrogen peroxide, fluorescence, pH, conductivity, extinction, colour, etc.
The device according to the invention provides the following advantages. The body of the stirrer may be easily taken out, as well as the other removably fixed members provided with immobilized enzyme. Thus they may be easily changed or renewed, as eventually it may be desired that the outer layer of fixed enzymatic material be changed without changing the characteristics of the sensor used for tracing the process of the reaction. The mixer or other object with the fixed enzyme or foil with fixed enzym-atic material for fixing onto the surface of the mixer or removable object may be prepared for stock and kept, without changing their activity, for several months.
8S~
By changing the mixer with enzymatic ~aterial immobilized on its surface for a mixer with another enzym-atic material fixed on its surface, one may analyse another substrate in the same reactor by means of the same sensor.
In a single sam~le, in the same reactor with a single sensor there may be determined, by a change of mixer with the respective immobilized enzymes, several substances, e.g. glucose in blood plasma with immobilized glucose-oxidase, by a change for an object with immobilized lactate-oxidase: lactic acid, and uric acid by a change for an object with immobilized urate-oxidase, all without changing the volume of the reaction tank or sensor.
By changing revolutions of the mixer or by changing the viscosity of the reaction mixture by adding substances affecting viscosity, e.g. of carboxymethylcellulose or poly-vinyl alcohol, one may control th~ effect of the outer diffusion on the rate of change of the traced quantity, and in this way one may change the calibration scope of the analyser. One achieves the ~one of the control function of the external diffusion of the substrate, if the concen-tration of the substrate on the surface of the fixed enzyme is kept at a value going near to zero, in any case a value lower than 3~ of the value of the initial concentration of the substrate in the sample. This zone may be achieved by various processes, e.g. by decressing the mixer revo-lutions,which provides an increased thickness of a liquid film on the surface of the layer with enzymatic material by increasing the sample viscosity of the tested liquid, which also provides a thickening of the liquid film and more over the diffusivity of the substrate decreases; or by increasing the activity of the immobilized enzyme. If one uses the device for controlling the external diffusion of the determined substance, other advantages may come in question:
~Z~8'~9 ~ simple mathematical description of the rate of change of the analysed quantity in dependence upon time, may be represented for a closed reactor by an exponential dependence of the value of the analyzed quantity, e.g. of oxygen concentration, vs time. This character of the dependence is the same, for any enzyme used or any concen-tration of any substance to be analyzed. A micro-computer, may be advantageously utilized for recognizing the signal because softwear equipment is simple and identical for a lot of instruments for determining various substrates.
A calibration dependence, residing in the value of the initial rate of change or the analysed quantity, e.g. rate of change of oxygen concentration, is then linear, but the gradient of the linear dependence is not dependent upon the activity of the enzyme; of course, if the concentration or activity of the enzyme is sufficiently high. The activity of the enzyme determines only the range, in which the calibration dependence is linear. The range of linear-ity decreases in dependence upon the decreasing activity 20 of the enzyme. A long-time stability of the device, viz.
of its calibration, may be achieved by applying a high concentration of the fixed enzyme. In this way any decrease of the activity does not affect the calibration, if for example the enzyme is able to keep up the concen-tration of the determined substrate on the surface of theenzymatic layer in the value near to zero at the upper limit of the range of the device. The increase of vis-cosity of the solution, or the decrease of mixer revolutions may provide an enlarging of the linear zone of calibration.
The calibration dependence is not sensible to mechanical shock. The mixer or the said takable out parts may be taken out of the reactor, taken in hand and put again into the reactor without changing the calibration of the device.
In order that the invention may be clearly under-121~3Z89 stood and readily carried into effect, a preferred embodi-ment thereof is, by way of example, hereinafter more fully described and illustrated in the accompanying drawings, in which: Figure 1 shows a schematic sketch of the device for carrying out the method, and Figure 2 shows a calibration curve.
Example:
A device for performing the method according to the invention is shown in Fig. 1. It consists of a tempering or temperature jacket 1, oxygen probe 2, the sensor of which is separated from the sample in the reactor 3 by means of a membrane 4 permeable to oxygen. Inside the tempering jacket 1, there is situated a magnetic stirrer 5, onto which a foil 7 with immobilized enzymes is fixed by means of a ring 8. The magnetic stirrer 5 is driven by means of a magnet 6. Directly on the polyacryl-amide surface of the magnetic stirrer 5 or on the foil 7 of the same material, as it is shown in Fig. 1, a mixture of glucose-oxidase and catalase in a total quantity of 20 international units is immobilized by means of any known method, e.g. by means of cyanuric chloride or carbodiimide or by combination of an enzyme with a suitable inert protei~
~nd glutaraldehyde. ~ sample - solution of buffer ~ith pH 6,6 comprising glucose, the quantity of which is to be determined, was put into the reactor. In the table there is in the first column the initial gradient of dependence of the signal of the oxygen probe 2 in any units, in the column 2 the initial concentration of glucose in the reaction mixture, in milimvls, and in the column 3 there is the number of micromols of the original solution. The table sho~s evidently that there was- achieved a wide zone of linearity of the calibration curve in the range 0 up to 0,7407 mM of the initial concentration of glucose. The _ g _ :~Z18~39 respective calibration curve is shown in Fig. 2.
Enzymes were fixed on a nylon mesh having size of eyes 50 um of area limited by a ring 8 of dia 10 mm.
Onto this area there were applied approx. 20 international units of crude glucose-oxidase comprising catalase as well.
The preparation of the solution for fixing glucose-oxidase onto the nylon mesh is carried out as follows: 100 ul of the mixture is to be prepared: 5 mg of glucose-oxidase dissolved in 50 ul of water, one adds 50 ul of 10% of beef albumin and 20 ml of 2% glutaraldehyde in 0,5 M of phosphate buffer pH 6,6. On the mesh into the ring 8 of dia 10 mm there is applied 15 up to 20 ul of this mixture and one let it dry up.
:12~ 8'~
T A B L E
Initial gradient Initial concentrat- ul of the ori-of dependence of ion of glucose in ginal solution the oxygen probe the reaction mixture of glucose 0.4 0.0185 1.25 1.0 0.0481 3,25 1.6 0.0741 5
2.35; 2.4+ Oollll 7,5 10 3.1; 3.2+ 0.1481 10 4.8 0.222 15 6.4 0.2963 20 8.5 0.3703 25 9.9 0.444 30 13.4 0.5926 40 16.1; 16.5* 0~7407 50 18.7 0.8888 60 24.4 1.481 100 ~/ repeated determination after 1 day The method according to the invention provides all advantages of hitherto processes, applying homo-genous solutions of enzymes, i.e. a quick response of the sensor, and at the same time the advantage of the immobilization of the enzyme, viz,its low consumption.
, . ,
, . ,
Claims (7)
1. A method for performing and analysing enzymatic reactions making use of immobilised enzymes, characterized in that the enzymatic reaction or reactions are performed in a reactor wherein the enzymatic material is immobilized on the surface of a removable stirrer and/or on the surface of one or more other removably fixed members, the stirrers and said members being disposed inside the reactor separately from the sensor of a measuring device and in a quantity sufficient to maintain the concentration of the tested substance on the surface of the enzymatic material during a plurality of measurements at from o to 3%, with respect to the concentration of the original solution.
2. A method as defined in claim 1, wherein the liquid reaction mixture includes a viscosity increasing substance.
3. A method as defined in claim 2, wherein the viscosity increasing substance is selected from the group consisting of carboxymethyl cellulose, polyvinyl alcohols and mixtures thereof.
4. A device for carrying out the method as defined in claim 1, comprising a reactor provided with said stirrer on the surface of which a layer of enzymatic material is immobilized.
5. A device as defined in claim 4, wherein the stirrer is a magnetic stirrer.
6. A device as defined in claim 1, comprising a reactor, inside which there is disposed said one or more removably fixed members, said members being provided with a layer of immobilized enzymatic material.
7. A method for performing and analysing enzymatic reactions making use of immobilized enzymes characterized in that the enzymatic reaction or reactions are perfomed in a reactor wherein, the enzymatic material is immobilized on a surface in the reactor which is separate from the sensor of a measuring device and in a quantity sufficient to maintain the concentration of the tested substance on the surface of the enzymatic material during a plurality of measurements at from 0 to 3% with respect to the concentration of the initial solution.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS835655A CS238261B1 (en) | 1983-07-28 | 1983-07-28 | Enzymatic reactions execution and tracing method and device for application of this method |
| CSPV5655-83 | 1983-07-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1218289A true CA1218289A (en) | 1987-02-24 |
Family
ID=5401824
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000459834A Expired CA1218289A (en) | 1983-07-28 | 1984-07-27 | Method for performing and tracing enzymatic reactions and a device for carrying out the said method |
Country Status (8)
| Country | Link |
|---|---|
| JP (1) | JPS6091996A (en) |
| AT (1) | AT389317B (en) |
| CA (1) | CA1218289A (en) |
| CS (1) | CS238261B1 (en) |
| DE (1) | DE3427444A1 (en) |
| FR (1) | FR2549852B3 (en) |
| GB (1) | GB2145815B (en) |
| IT (1) | IT1205647B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0884392B1 (en) * | 1997-06-03 | 2002-12-18 | Matsushita Electric Industrial Co., Ltd. | Cholesterol sensor |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1568111A (en) * | 1975-07-22 | 1980-05-29 | Phosphor Prod Co Ltd | Electroluminescent devices |
| SE396819B (en) * | 1975-12-31 | 1977-10-03 | Gambro Ab | METHOD AND DEVICE FOR DETERMINING THE CONCENTRATION OF A LOW MOLECULAR ASSOCIATION IN A COMPLEX MEDIUM BY DIALYSIS |
| DE2642232C3 (en) * | 1976-09-20 | 1980-05-14 | Boehringer Mannheim Gmbh, 6800 Mannheim | Method and device for the continuous determination of the concentration of an enzyme substrate |
| GB1571466A (en) * | 1976-12-14 | 1980-07-16 | Univ California | Assay mehtod |
| JPS6029B2 (en) * | 1978-09-06 | 1985-01-05 | 藤沢薬品工業株式会社 | Immobilized enzyme column |
| IT1113361B (en) * | 1979-05-08 | 1986-01-20 | Italfarmaco Spa | FLOW REACTOR WITH ENZYMES IMMOBILIZED ON SOLID FLAT SURFACES |
| JPS56164797A (en) * | 1980-05-21 | 1981-12-17 | Toyo Jozo Co Ltd | Improved method of determining components in samples |
| JPS5718979A (en) * | 1980-07-09 | 1982-01-30 | Olympus Optical Co Ltd | Measuring container for use in analysis with immobilized enzyme |
| IT1137262B (en) * | 1981-06-26 | 1986-09-03 | Erba Farmitalia | AUTOMATIC METHOD ON CONTINUOUS FLOW FOR THE DOSAGE OF CREATININE WITH IMMATIZED DESIMIDASE CREATININ |
-
1983
- 1983-07-28 CS CS835655A patent/CS238261B1/en unknown
-
1984
- 1984-07-25 DE DE19843427444 patent/DE3427444A1/en active Granted
- 1984-07-27 IT IT8422105A patent/IT1205647B/en active
- 1984-07-27 CA CA000459834A patent/CA1218289A/en not_active Expired
- 1984-07-27 JP JP59155675A patent/JPS6091996A/en active Pending
- 1984-07-27 AT AT0243484A patent/AT389317B/en not_active IP Right Cessation
- 1984-07-27 FR FR8411999A patent/FR2549852B3/en not_active Expired
- 1984-07-27 GB GB08419285A patent/GB2145815B/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| ATA243484A (en) | 1989-04-15 |
| FR2549852A1 (en) | 1985-02-01 |
| DE3427444C2 (en) | 1989-09-07 |
| GB2145815B (en) | 1987-07-29 |
| GB2145815A (en) | 1985-04-03 |
| IT1205647B (en) | 1989-03-23 |
| DE3427444A1 (en) | 1985-02-07 |
| IT8422105A0 (en) | 1984-07-27 |
| CS238261B1 (en) | 1985-11-13 |
| FR2549852B3 (en) | 1985-11-22 |
| JPS6091996A (en) | 1985-05-23 |
| AT389317B (en) | 1989-11-27 |
| GB8419285D0 (en) | 1984-08-30 |
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