Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
21~3618
BIOSENSOR FOR DETECTION OF AND FOR DISTINGUISHING BETWEEN
INHIBITORS OF CHOLINESTERASES
FIELD OF THE ART
The invention relates to the analysis of biologically very
effective organophosphorous and carbamate inhibitors of
cholinesterases by a simple biosensor. The invention makes
possible to use the sensitive biochemical cholinesterase
reaction to detect and distinguish between the biologically
extremely effective organophosphates and carbamates that are
permanently used in the field of the industrial chemistry,
agrochemistry and in the military sector.
STATE OF THE ART
The biochemical reaction of cholinesters is a-very sensitive
and selective method that is widely used for a long time in
a number of modifications and applications to detect and
determine a numerous group of agents that are
cholinesterases inhibitors [AMMON, R. - VOSS, G., Pflugers,
Arch. ges. Physiol., 235 (1935), p. 393; MICHEL, 0., H., J.
Lab. clin. Med., 34 (1949), p. 1564]. Technical means of
various design used to detect and determine cholinesterases
inhibitors, starting with the complicated automatic
atmosphere and water analysers, continuing with more simple
designs, e.g. tube detectors and ending with the most simple
but therefore more used detection test papers, are based on
the cholinesterase reaction. [FRANKE, S.: Lehrbuch der
Militarchemie, Militarverlag DDR, part II, Berlin 1977, p.
481; U.S. patent No. 4.324,858; ~O 8504424]. Regardless of
AMENDEO StlEEr
_ - 2 _ 21~ 361
the total complexity of the design and financial costs all
categories of means maintain a similar sensibility and
selectivity owing to the same reaction principle.
Consequently, in the absolute majority of cases the most
simple and therefore also the cheapest design, i.e. the
detection paper is preferred. The individual detection
papers differ in two ways. At first, in the used substrate
and consequently in the relevant indication reaction. This
part of the cholinesterase reaction materialization in the
test paper is not essential as it cannot have an influence
on the applicability for determinations in water and
atmosphere, but it can influence the determination
sensitivity. The basic problem is the method how to fix and
stabilize the enzyme. Some existing methods are based on the
enzyme adsorption in the filtration paper [AO CSFR 269267].
Another solution of this problem is to immobilize
cholinesterase in the carrier by a chemical bond, what is
accompanied by a significant hydrolytic activity loss of the
enzyme. Additionally, it requires to use a highly purified
and therefore expensive enzyme for the immobilization,
whereby prolongation of the enzyme paper expiration time is
not very high [RO 105190].
The nearest disclosed design to the one according to this
invention is to prepare the enzyme detection paper by
immobilization of cholinesterase in a special
chromatographic paper with ion exchange function in mixture
with dextrane gel and to make indication by a chromogenic
substrate [~O 85/04423]. However, in this case the required
sensitivity of the detection paper was not o~tained in case
of increased storage time. In spite of a great number of
various modifications relating to the method of evaluation
of the used enzyme, substrate, various environments or to
modification of the other workmanship conditions, all such
modifications are limited by the fact that the biochemical
cholinesterase reaction is a group reaction for a very
AMEND~D SHEET
2 1 J~ 3 6 1 8 r
numerous group of the cholinesterases inhibitors. The most
important of them are the organophosphorous compounds and
the carbamates. To make possible to identify what inhibitor
is present, it is necessary to carry out some detection
reactions or physicaZ chemistry measurements. However, this
requires a greater amount of inhibitor or by several orders
higher inhibitor concentration in the sample solution than
as it is necessary in case of the very sensitive biochemical
cholinesterase method. In such case it is necessary to carry
out difficult, complex and lengthy sample concentration
preceding the proper chemical or physical chemistry
identification. Such methods with the exception of some more
simple separation and identification techniques, as is the
thin-layer chromatography, are not only time consuming but
also require further operations connected with performing
the analytical detection reactions or with the use of
expensive instruments, that can not be operated in the
field. Moreover, considering the extreme biological
effectiveness~ a number of the organophosphorous and
carbamate cholinesterases inhibitors constitutes also
a substantial risk with regard to labour safety if we
consider the duration of the necessary operations with
concentrated solution of the compound which is to be
identified.
The nearest disclosed solution of the analysis method with
regard to this invention is the method of the
organophosphorous inhibitors identification which is based
on the formation of two types of complexes with metal ions
and the detection by ~he cholinesterase reaction
[CEBOTARJOV, 0., V.: Vojskovaja indikacia, Izdatelstvo
akademii Moscow 1978] and the method of distinguishing
between the organic inhibitors by the cholinesterase
reaction after cholinesterase was modified by metal ions.
The disadvantages mentioned consisting in the group nature
of the biochemical cholinesterase reaction require that
AMENDED SIIEE~
- ~ 4 ~ 219 361 8 ' "'"
laborious and lengthy inhibitor sample concentration is
necessary and that expensive and complicated instruments are
to be used for identification. The above disadvantages are
removed by detection of organophosphorous and carbamate
cholinesterases inhibitors according to this invention.
THE EMBODIMENT OF THE INVENTION
The invention consists in a biosensor used to detect and
identify cholinesterases inhibitors in atmosphere, water,
foodstuffs, soil, surfaces and extracts from samples. The
biosensor in the form of test strips consists of a cellulose
cloth zone made of cotton fibres containing immobilized
cholinesterase, of a colour standard and of an inert carrier
zone, e.g. of a filter paper with substrate and chromogenic
agent. During the cholinesterase inhibitors detection,
e. g. the acetylthiocholiniodide substrate and the 5,5'-di-
thiobis(2-nitrobenzoic) acid as chromogenic agent, are
transferred to a wet cellulose cloth cont~;n;ng immobilized
cholinesterase exposed in a controlled environment, by
pressing filter paper cont~;n;ng them towards it. The
chromogenic agent reacts by colour change to products of the
substrate hydrolysis. The hydrolytic activity of the
immobilized cholinestarase is visually evaluated by
comparison with the colour standard as a change of the
cellulose cloth colour. Application of this biosensor to
distinguishing between inhibitors consists in that in case
of cholinesterases immobilized on cellulose cloth, after
inhibition by organophosphate or carbamate inhibitor, the
hydrolytic activity is restored by induced reactivation by
quaternary aldoximes, e.g. by l-(2-hydroxyiminomethylpyri-
dinum)-3-(4-carba-moylpyridinum-2-oxapropan)dichloride
(further only HI-6) and N,N'-tri-methylenbis(4-pyridinium-
adoxim)dichloride (further only TMB-4) or by spontaneous
reactivation by water. Simultaneously, the samples of
cholinesterase immobilized on cellulose cloth of the
AMENDED S~IEET
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biosensor are exposed in water solution of inhibitor and in
the water solution of inhibitor with the addition of ionic
compounds, e.g. of natrium, calcium or ammonium chloride. By
evaluation of the hydrolytic activity of the reactivated
cholinesterase and of the activity of the cholinesterase
exposed in the inhibitor solution and in the inhibitor
solution with the added sodium, calcium or ammonium
chloride, a characteristic difference in activity is
indicated that makes possible to identify which inhibitor it
is.
In comparison to the prior art the present invention is
distinguished by the carrier nature and carrier treatment
that makes an easy immobilization of enzyme and further
by possibility of distinguishing inhibitors by the
biosensor.
By the use of 100 ~ cotton cloth for immobilization of
cholinesterase without further treatment and additives
a composite material having new biochemical, mechanical and
optical characteristics is formed.
The above can be documented by the following
characteristics:
The used carrier - the cotton cloth can be used in a simple
immobilization of enzymes of different etiology and quality
and the obtained cellulose - cholinesterase composite is
very stable.
The used carrier containing immobilized enzyme is of such
superior mechanical properties that it allows repeated, up
to 60 times, use of the detection cloth, long time
exposition in running water or long time exposition in
untreated complex samples, e.g. in food homogenizates.
U- a S~
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The detection cloth makes possible to wash out perfectly the
co-extracted impurities and colours that would interfere
with the visual evaluation of the reaction course.
All said above is not possible in case of the disclosed
carriers.
Methods of simple and effective immobilization of
cholinesterases of various etiology and purity giving
products stable at usual and elevated (up to 60 ~C)
temperature are not known.
Research of the inhibited enzymes is in reality limited by
the possibility to isolate them from the analyzed
environment. An effective isolation from the environment is
ensured by preparation a stable enzyme composite cotton
cloth - cholinesterase and the difference in structure of
the isolated enzyme-inhibitor complexes can by studied
individually.
AMEN~)ED SHEET
7 ~ 2 1 4 3 6 1 8
EXAMPLES
Example 1
Preparation of acetylcholinesterase solution
The prepared acetylcholinesterase is used to prepare a solu-
tion having specific activity 7 to 15 nkat/ml by dissolving
it in a solution buffered of pH 5 to 9.
Example 2
Modification of cellulose cloth.
A cellulose cloth woven from cotton fibres is modified by
alkaline oxidative bleaching by boiling the cloth in
a solution cont~inlng 7.9 g/l of hydrogen peroxide (35 %),
0.2 g/l of magnesium chloride, 0.2 g/l of non-ionogenic
tenside SLOVASOL SF-10, 2.1 g/l of sodium hydroxide and 2.5
g/l of water glass.
Example 3
Immobilization of acetylcholinesterase on bleached cellulose
cloth
The cloth modified according to Example 2 is washed by water
and dried at 50 C and thereafter immersed in the solution
prepared according to Example 1 for 30 minutes. Thereafter,
it is taken out of the solution and dried at 20 C.
Example 4
Preparation of the carrier with substrate and chromogenic
agent
A filter paper is immersed in a solution of 0.5 % of acetyl-
thiocholiniodide and 0.15 % of 5,5'-dithio-bis(2-nitro-
benzoic) acid in ethanol for 10 minutes. Thereafter, thepaper is dried at 20 C.
AMENDED ~!!EET
21:4,~618 ' - '
Example 5
Detection of inhibitors in water and in samples of water
homogenizates
The detection cloth prepared according to Example 3 is
immersed into water or water extract for 30 minutes.
Thereafter, the cloth is taken out and for 1 minute paper
with substrate and chromogenic agent prepared according to
Example 4 is pressed towards it. After the paper is removed
the cloth is yellow coloured in case of acetylcholinesterase
inhibitors absence. Unchanged colour of the cloth is an
evidence that inhibitors are present.
Sensitivity to organophosphate and carbamate insecticides
detection in water and water extracts from foodstuffs
Insecticide Detection Limit (mg/l)
CARBOFURAN 2.10-2
METHOMYL 1.10-
DICHLORVOS 1.10-1
MEVINPHOS 1.10-2
PARATHION - 2.10-2
~ETHYLPARATHION 4.10-2
Example 6
Inhibitors detection in air
Detection cloth prepared according to example 3 is made wet
by water and left in a controlled atmosphere minimally 1
minute. Thereafter, paper with substrate and chromogenic
agent prepared according to Example 4 is pressed towards the
cloth. After the paper is removed the cloth is coloured
yellow if inhibitors of cholinesterase are not present. An
evidence of inhibitors is the unchanged colour of the
textile.
AM~ND'a ~'
2 1 ~
Detection sensitivity to nerve agents detection in
atmosphere
nerve agent detection limit (mg/l)
after exposition
2 min 20 min
isopropylmethylfluorophosohonate 5 7
SARIN GB 1.10- 4.10-
pinakolylmethylfluorophosohonate 6 7
SOMAN GD 8.10- 2.10-
0-etthyl-s-(2-diisopropylamino-
ethyl)methylthiophosphonate 5 7
AGENT VX 5.10- 5.10-
2-dimethylamionoethyldimethyl-
amidofluorophosphate
ACENT GP 5.10-6 2.10-7
ethyldimethylamidocyanophosphate 5
TABUN GA 8.10- 1.10-6
cyclohexylmethylfluorophossphonate 6 7
CYKLOSIN GF 3.10- 1.10-
Example 7 --
Use of biosensor to distinguish between GD and GF compounds
Cloth samples containing the immobilized acetylcholin-
esterase inhibited to 100% by 2 to 5 minutes of incubation
in a solution containing organophosphate inhibitor are reac-
tivated for 5 minutes in 0.02 mg/ml of ~I-6 and 0.05 mg/ml
of TMB-4. After reactivation, hydrolytic activity of the
enzyme in both samples containing inhibited cholinesterase
is evaluated. If the hydrolytic activity of the inhibited
enzyme was restored only by reactivation in 0.02 mg/ml of
HI-6, the solution contains the GD or GF compound. The
reactivation is repeated after the inhibition of a sample of
immobilized acetylcholinesterase by 30 minutes of
incubation. ~hen the enzyme hydrolytic activity is restored
AMENOED SHE~T
2143618
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by the reactivation the solution contains the GF compound.
Example 8
Use of biosensor to distinguish between VX and GB compounds
Cloth samples containing the immobilized acetylcholin-
esterase inhibited to 100% by 2 to 5 minutes of incubation
in the solution containing organophosphate inhibitor are
reactivated for 5 minutes in 0.02 mg/ml of HI-6 and 0.05
mg/ml of TMB-4. After reactivation, the enzyme hydrolytic
activity in both samples of inhibited cholinesterase is
evalua~ed. If the hydrolytic activity of the inhibited
enzyme was restored by reactivation in both reactivators the
solution contains the compound VX or GB. The solution is
diluted ten-times and divided to two parts. To one part of
the diluted solution natrium chloride is added in surplus
and in both solutions samples of immobilized acetylcholin-
esterase are simultaneously incubated for 2, 5, 10, and 15
minutes till different hydrolytic activities of samples are
reached. In case of an increase of the sample hydrolytic
activity which was incubated in the solution with natrium
chloride in comparison to the activity of the sample
incubated in the solution without natrium chloride the
analyzed sample contains the VX compound.
Example 9
Use of biosensor to distinguish between GA and GP compounds
Samples of the immobilized acetylcholinesterase inhibited to
100% by 2 to 5 minutes of incubation in the solution
containing organophosphate inhibitor are reactivated for 5
minutes in 0.02 mg/ml of HI-6 and 0.05 mg/ml of TMB-4. After
reactivation, the enzyme hydrolytic activity is evaluated in
both samples of inhibited cholinesterase. If the hydrolytic
AME~IDEDSHEET
- 11 219~618
activity of the inhibited enzyme is not restored in both
reactivators the sample contains solution of the GA or GP
compound. The solution will be ten-times diluted and divided
to two parts. Natrium chloride is added in surplus to one
part of the diluted solution and simultaneously in both
solutions the samples of immobilized acetylcholinesterase
will be incubated for 2, 5, 10 and 15 minutes till
a different hydrolytic sample activity is obtained. In case
the hydrolytic activity of the sample incubated in the
solvent with natrium chloride is higher than that of the
solution without natrium chloride, the analyzed sample
contains the GP compound.
Example 10
Use of biosensor to distinguish organophosphorous and
carbamate insecticides
.
Samples of the immobilized acetylcholinesterase inhibited to
100% by 2 to S minutes of incubation in the solution
containing organophosphate or carbamate inhibitor are
reactivated for 5 minutes in 0.02 mg/ml of HI-6 and 0.05
mg/ml of TMB-4. After reactivation, the enzyme hydrolytic
activity in both samples of inhibited cholinesterase will be
evaluated. If hydrolytic activity of the inhibited enzyme is
not restored after 5 minutes of reactivation in both reacti-
vators, the sample is immersed for 30 to 120 minutes into
distilled water. If the hydrolytic activity is restored
after 60 minutes of reactivation in water, the solution
contains l-naftyl-~-methylcarbamate. If the hydrolytic
activity is restored after 120 minutes of reactivation in
water, the solution contains 2,3-dihydro-2,2-dimethylbenzo-
furane-7-yl-~-methylcarbamate.
A~ !DE~
21436I8 .
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Example 11
Biosensor use in identification of nerve paralyzing agents
Two samples of the detection cloth are again immersed into
the analyzed solution containing an unspecified nerve gas in
which the detecting cloth sample containing immobilized
acetylcholinesterase remained white after 2 minutes of
exposition. Thereafter, the samples are immersed into the
reactivator solution for S minutes, one sample into 0.05
mg.ml~l of TMB-4 solution and the other into 0.02 mg.ml~l of
the HI-6 solution. ~hen the cloth is removed from the
reactivators solutions three alternative possibilities can
be observed with regard to the cloth activity:
a) Both cloth samples remain white. In this case the
solution contains the compound GA or GP.
b) Both cloth samples become yellos. In this case the
solution contains the compound VX or GP.
c) The cloth reactivated in TMB-4 remains white. The cloth
which has been reactivated in HI-6 becomes yellow. In
this case the solution contains the compound GF or GD.
Identification of the individual compounds from the alterna-
tives a) or b) is carried out so that the solution is
diluted by water in ratio 1:1 and divided in two parts. NaCl
is added to one part so that a saturated solution or
a solution with surplus of undissolved NaCl is obtained.
Thereafter, inhibition in both solutions is compared. If the
cloth inhibited by the solution with NaCl becomes less
yellow and the cloth from the solution without NaCl remains
white, the solution contains the VX or GP compounds. In the
c) case it is possible to identify GF from GD so that
a sample of the detection cloth is exposed in the original
solution for 30 minutes. ~hen the exposition is inished the
AMEN~)~O SHE~
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cloth sample is immersed into the solution of ~I-6 for 5
minutes and if after reactivation the cloth becomes yellow
the solution contains GF. In case the cloth remains
inhibited even after the reactivation the solution contains
the GD compound.
The invention can be used in qualitative and quantitative
determination of organophosphorous and carbamate inhibitors
of cholinesterase. Such determination is carried out in many
industries and agriculture, but also by medical and
veterinary institutions. As a method of detection and
determination of organophosphorous and carbamate poisons the
modified cholinesterase reaction according to the invention
can be used also in the field of military chemistry if
chemical reconnaissance or tests are carried out, in civil
defense and state safety matters and in monitoring
environment contamination by inhibitors of cholinesterases.
The biosensor according to this invention is applicable in
testing contaminated samples of various etiology and
composition. Rapid field checking without usual laboratory
equipment is possible.
AM~NDED~E~
