Note: Descriptions are shown in the official language in which they were submitted.
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
-- 2 --
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.
-- 10 --
: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.
, . ,
