Title of Invention	MICROENCAPSULATED ENZYME BIOSENSOR FOR PESTICIDE
Abstract	The present invention relates to a microencapsulated enzyme biosensor device comprising plurality of electrodes including reference/counter electrode (2) and working electrode (1). Working electrode (1) comprises a loop (6) so as to ensure adhesion of polyacrylamide hydrogel, and the counter electrode (2) have relatively large area than the working electrode (1). The invention also relates to a method for producing immobilized micro- encapsulated with minimum loss of enzyme activity. The method comprises steps of spray drying of an emulsion to form encapsulated CAB enzyme powder and immobilizing the encapsulated enzyme powder by hydrogel being formed by bulk polymerization.

Title of Invention

MICROENCAPSULATED ENZYME BIOSENSOR FOR PESTICIDE

Abstract

The present invention relates to a microencapsulated enzyme biosensor device comprising plurality of electrodes including reference/counter electrode (2) and working electrode (1). Working electrode (1) comprises a loop (6) so as to ensure adhesion of polyacrylamide hydrogel, and the counter electrode (2) have relatively large area than the working electrode (1). The invention also relates to a method for producing immobilized micro- encapsulated with minimum loss of enzyme activity. The method comprises steps of spray drying of an emulsion to form encapsulated CAB enzyme powder and immobilizing the encapsulated enzyme powder by hydrogel being formed by bulk polymerization.

Full Text	FIELD OF INVENTION The present invention relates to an enzyme biosensor device. More particularly, the invention relates to an improved device for amperometric estimation of carbamate or organo-phosphorous based pesticides in a given liquid sample by way of improved immobilization of microencapsulated enzyme thereby enhancing the efficacy of the device. The invention also relates to a method for producing microencapsulated electrode for amperometric estimation of pesticides in a given liquid sample. BACKGROUND AND PRIOR ART In modern days the rampant use of organophosphorus-based pesticides is creating high risk of health hazards. Farmers all over the world use organophosphorus-based pesticides and insecticides extensively. These neurotoxic compounds are highly toxic because of their inhibitory effect on cholinesterase enzymes essential for the functioning of the central nervous system in humans and insects. As acetylcholinesterase (AChE) promotes the hydrolysis of natural neurotransmitter, acetylcholin (ACh), its inhibition terminates the propagation of the nerve impulse. In a recent US geological survey study, widespread presence of trace amounts of these pesticides was found in surface and ground waters across the US. A similar presence of the deadly organophosphorus could be expected in the water resources across India. So, there is a need for development of sensitive methods for the detection of cholinesterase inhibitors for both environmental monitoring and clinical diagnosis. Traditionally, pesticides residues could be measured by the methods of liquid chromatography-mass spectrometry (LC-MS), high performance liquid chromatography 2- (HPLC), immunoassay e.g. enzyme linked immuno sorbant assay (Elisa) etc. Highly sensitive HPLC or GC procedures were also developed for the analytical determination of these pesticides. However, these processes were not very effective, as the amount of pesticide could not be measured precisely. Prior art discuss about biosensor, screen-printed biosensor, electrical biosensor, receptor- based biosensor and spectroscopic methods for detection of chemical toxins. Most biosensor discussed in prior art consists of enzymes that are immobilized by means of covalent bonds. Immobilizing the enzymes by means of covalent bond reduces the enzyme activity and hence efficiency of biosensor. Immobilization is a process by which the biomaterials are held firmly on the transducer surface. The process has been widely used by researchers in biosensors. Some examples are adsorption, covalent bonding, cross-linking etc. The water-in-oil emulsions are produced with a particular emulsifier such that the aqueous phase is inside a thin layer of the organic phase (polymer). This was sprayed through a spray drier to form beads of polymer having the biomaterial inside it. US6406876 describes a method for detecting hazardous chemicals using biosensor. The biosensor consists of enzymes that are immobilized on or inside a porous or a non-porous support by covalent bonds. Covalent bonding of the enzyme with the support reduces enzyme activity. US5001048 describes electrical biosensor containing a biological receptor immobilized and stabilized in a protein film. In case of binding a biological receptor in a protein film the key mechanism of binding is covalent bonding. US4324858 describes immobilization of enzyme by impregnation into a porous material. The porous sheet, which has been used, is difficult to fix on the working electrode. Also the covalent bonding has been encouraged by using sulphonic, amine groups etc. 3 US5192507 discloses a method of immobilizing and stabilizing an active biological receptor in a polymeric film and receptor-based biosensor for determining an analyte of interest in a sample. In case of immobilization of enzyme in a polymeric film by materials capable of polymerizing and a polymerizing agent may result in covalent bonding of the enzyme in the polymeric film. US4411989 describes methods and devices for identifying substances (such as chemical warfare agents) and residual environmental pollutants (such as pesticides). These utilize the discovery that spectra of an uninhibited enzyme (e.g., a cholinesterase) can differ from spectra of the same enzyme, which has been complexed with the agent pollutant. Analysis by spectroscopic method has lower sensitivity. None of the prior art discussed above describe bulk polymerization technique for improved immobilization of microencapsulated enzyme in amperometric estimation of pesticides for a given liquid sample. Basic reaction for determination of pesticide : Enzyme AchE (acetycholine eaterase) hydrolyzes acetylcholine and acetic acid The choline is oxidized by oxygen catalyzed by the enzyme ChO (choline oxidase) and H2O2 is produced in the process. Since H2O2 produced has a very low concentration, it may not be detected by direct electrochemical route. However good signals can be obtained for a low level (ppb) using TMB and HRP. The TMB is rapidly oxidized by H2O2 in presence of HRP added to the system. Due to redox reaction there will be exchange of electrons at the working electrode. Thus amperometry can be done for indirect detection of H2O2. Acetylcholine esterase is partly deactivated by organo-phosphorous (OP) based pesticides 5 due to formation of permanent covalent bond. Thus there is a need for providing a device for amperometric estimation of pesticides in a given liquid sample by way of improved immobilization of microencapsulated enzyme thereby enhancing the efficacy of the device. There is also a need to provide for a method for producing microencapsulated electrode for amperometric estimation of pesticides in a given liquid sample. Thus the present inventors have found that amperometric estimation of carbamate or organo-phosphorous based pesticides in a given liquid sample can be achieved by way of improved immobilization of microencapsulated enzyme by bulk polymerization thereby providing enhancement in overall efficiency of the device. OBJECTS OF INVENTION Accordingly, one object of the present invention is to provide a device for amperometric estimation of pesticides in a given liquid sample by way of improved immobilization of microencapsulated enzyme. Another object of the invention is to provide a method for producing microencapsulated electrode for use in amperometric estimation of pesticides in a given liquid sample. Another object of the present invention is to provide bulk polymerization. Yet another object of the invention is to provide a simple, efficient and cost effective method for amperometric estimation of pesticides. SUMMARY OF INVENTION According to one aspect of the present invention there is provided a microencapsulated enzyme biosensor device for amperometric estimation of carbamate and/or an organophosphorus based pesticides in a given sample, said device comprising : 6 plurality of electrodes including reference/counter electrode and working electrode, wherein said working electrode comprising a loop so as to ensure adhesion of polyacrylamide hydrogel, and wherein said counter electrode having relatively large area than the working electrode. Another aspect of the present invention is to provide a method for producing immobilized micro-encapsulated with minimum loss of enzyme activity, said method comprising steps of: spray drying of an emulsion comprising predetermined quantity of acetylcholine esterase enzyme dissolved in phosphate buffer forming an aqueous phase and organic phase prepared with cellulose acetate butyrate (CAB) dissolved in dichloromethane (DCM) to form encapsulated CAB enzyme powder; and further immobilizing said encapsulated enzyme powder by hydrogel being formed by bulk polymerization of a mediator selectively comprising acrylamide monomer, N,N methylene-bis-acrylamide (BIS) and glutaraldehyde and an initiator being selectively potassium peroxodisulphate. DETAILED DESCRIPTION OF INVENTION The present invention is directed to provide a microencapsulated enzyme biosensor for amperometric estimation of pesticides. The biosensor consists of two electrodes namely reference/counter electrode made of silver wire and working electrode made of platinum wire. However, this does not restrict the scope of the present invention. Graphite, glassy carbon, gold, rhodium etc. can be used as working electrode. But platinum is the best choice due to its inertness and selectivity. Moreover platinum electrode can operate at a much lower applied potential compared to that of other electrodes. At higher applied potential other electrodes also respond to all other electro-active components in the sample. Silver/silver chloride system or calomel electrode may be used as true reference 7 electrode. Silver may be used as pseudo reference electrode to reduce the size of the three electrode assembly. Calomel electrode is not preferred due to environmental hazard. All the wires are coated with some insulation selected from PVC tubing, glass capillary, epoxy coating etc. and the like. In case of glass capillary or PVC tubing the ends are sealed by heating or by epoxy. These coated electrodes are set on a polyester matrix using adhesives. Polymer or glass separators are placed to avoid short circuit between electrodes. Loops are made at the end of reference and working electrode. The loop at the terminal of the working electrode ensures adhesion of polyacrylamide hydrogel. In one preferred embodiment of the invention the terminal of the counter electrode has been coiled to ensure larger area compared to that of the working electrode. Preparation of microencapsulated enzyme: Microencapsulated enzyme is produced by spray drying technique. A mini spray drier (model no. B191, Buchi, Germany) is used for this purpose with an airflow rate-800 ml/s; pressure 500 KN/m2 and temperature 45 °C. Measured quantity of acetylcholine esterase enzyme is dissolved in phosphate buffer pH 7.4 to form the aqueous phase. The organic phase is prepared with CAB dissolved in dichloromethane (DCM). These two were then emulsified with SPAN-85. The emulsion then fed into the spray dryer in co current mode. The encapsulated CAB enzyme powder is separated by the cyclone separator and collected. Immobilization of the sensing element on the working electrode: Microencapsulated AchE (0.03U), ChO (0.03U), HRP (10U) and TMB (0.1 mg) are immobilized directly on the tip of a Platinum wire (working electrode) by bulk polymerization of acrylamide monomer, BIS (0 to 10% w/w of amount of monomer, preferably 4%) and glutaraldehyde (0 to 10% v/w of amount of monomer, preferably 1%) as crosslinkers, Ferrocene carboxylic acid (0.001 to 0.1% w/w of amount of monomer, 8 preferably 0.01%) as mediator and potassium peroxodisulphate (0.1 to 20% w/w of amount of monomer, preferably 5%) as initiator. Bi-functional molecules containing -NH2,-CHO,-COOH as functional groups can be used as cross linkers. N,N methylene-bis-acrylamide (BIS) is preferred as it forms irreversible gel. Increase in concentration of BIS makes the gel brittle. On the other hand glutaraldehyde provides flexibility to the gel. Thus the simultaneous uses of the two cross linkers helped preparation of a stable and workable gel with proper cross-link density. Mediators like ferrocene, ferrocene carboxylic acid, potassium ferricyanide etc. can be used. In the present invention ferrocene carboxylic acid is preferred to ferrocene due to its solubility in water. Potassium ferricyanide is not preferred due to its inhibitory effect on polymerization. Initiators that generate free radicals can be used. Thus initiators like benzoyl peroxide and ammonium peroxodisulphate can be used. The potassium perdisulphate is preferred as it ensures faster polymerization at room temperature. The biosensor device as described above can be used for estimation of any hazardous chemicals that can deactivate the enzyme Acetylcholine esterase. Estimation of pesticide: The microencapsulated polymer electrode was dipped in liquid sample containing pesticide for 15 minutes. It was then washed with buffer and dried in air. The treated microencapsulated polymer electrode and silver electrode were partly dipped into phosphate buffer containing 3mM KCL, pH 7.4 and amperometry was done at -0.25V. The microencapsulated polymer electrode acts as a working electrode and the silver electrode was used as pseudo reference electrode and counter electrode. After equilibrium measured quantity of Acetylcholine chloride solution was added. Reactions for the estimation of pesticides: The enzyme Acetylcholine esterase hydrolyzes Acetylcholine to choline and acetic acid. 9 Since H2O2 produced has a very low concentration, it may not be detected by direct electrochemical route. However, good signals are obtained for a low level (ppb) using TMB and HRP. The TMB is rapidly oxidized by H2O2 in presence of HRP added to the system. Due to redox reaction there will be exchange of electrons at the working electrode. BRIEF DESCRIPTION OF ACCOMPANYING FIGURES Figure 1 illustrates a schematic diagram of the micro encapsulated biosensor of the present invention. Figure 2 illustrates calibration curve for organophosphorous based pesticide. 10 DETAILED DESCRIPTION OF ACCOMPANYING FIGURES As illustrated in figure 1 the biosensor comprise working platinum electrode (1) which is placed parallel to reference/counter silver electrode (2) The working electrode (1) as well as the reference electrode (2) are coated with insulation coating (3). Since the biosensor is constructed as described above, there can be obtained a biosensor in which the respective electrodes are surely separated. This separator (4) is placed to avoid short circuit between electrodes (1) and (2). Two electrodes (1) and (2) are set on a polyester matrix (5). Loop (6) is made at the end of working electrode (1). Loop (6) is such that it ensures adhesion of microencapsulated enzyme acrylamide polymer hydrogel (7). In figure 2 calibration curve for organophosphorous based pesticide is illustrated. With increase in concentration of the pesticide there will be more deactivation of the enzyme acetylcholine esterase. Thus at a specified time interval less choline will be produced by oxidation of the substrate acetylcholine chloride due to the catalytic effect of the deactivated enzyme AchE. The amount of hydrogen peroxide produced by the action of ChO is directly proportional to the amount of choline produced by the earlier reaction. Again the amperometric response of TMB is proportional to the concentration of hydrogen peroxide. The graph shows that there is a linear decrease in rate of change in current with increase in concentration of pesticide. To check whether the sensors were able to give reproducible results, each experiment was repeated thrice. The error bars based on standard deviation value indicate that the responses are clustered closely around the mean i.e., the responses are reproducible. 11 WE CLAIM 1. A microencapsulated enzyme biosensor device for amperometric estimation of carbamate and/or an organophosphorus based pesticides in a given sample, said device comprising: plurality of electrodes including reference/counter electrode and working electrode, wherein said working electrode comprising a loop so as to ensure adhesion of polyacrylamide hydrogel, and wherein said counter electrode having relatively large area than the working electrode. 2. Device as claimed in claim 1 wherein the counter electrode includes a silver electrode adapted to be used as pseudo reference electrode. 3. Device as claimed in claim 2 wherein the counter electrode comprises insulation coating selected from PVC tubing, glass capillary, epoxy coating and the like. 4. Device as claimed in claim 1 wherein the working electrode is a platinum electrode. 5. Device as claimed in claim 4 wherein the platinum electrode comprises insulation coating selected from PVC tubing, glass capillary, epoxy coating and the like 6. Device as claimed in any preceding claims further comprising polymer or glass separator. 7. A method for producing immobilized micro-encapsulated with minimum loss of enzyme activity, said method comprising steps of: 12 spray drying of an emulsion comprising predetermined quantity of acetylcholine esterase enzyme dissolved in phosphate buffer forming an aqueous phase and organic phase prepared with cellulose acetate butyrate (CAB) dissolved in dichloromethane (DCM) to form encapsulated CAB enzyme powder; and further immobilizing said encapsulated enzyme powder by hydrogel being formed by bulk polymerization of a mediator selectively comprising acrylamide monomer, N,N methylene-bis-acrylamide (BIS) and glutaraldehyde and an initiator being selectively potassium peroxodisulphate. I3 8. Method as claimed in claim 7 wherein the phosphate buffer has a pH of 7.4. 9. Method as claimed in claim 7 wherein N,N methylene-bis-acrylamide (BIS) is present in the range of 0 to 10% w/w, preferably 4% of the amount of monomer. 10. Method as claimed in claim 7 wherein glutaraldehyde is present in the range of 0 to 10% v/w, preferably 1% of the amount of monomer. 11. Method as claimed in claim 7 wherein ferrocene carboxylic acid is further present in the range of 0.001 to 0.1% w/w, preferably 0.01% of amount of monomer. 12. Method as claimed in claim 7 wherein potassium peroxodisulphate is present in the range of 0.1 to 20% w/w, preferably 5% of amount of monomer. The present invention relates to a microencapsulated enzyme biosensor device comprising plurality of electrodes including reference/counter electrode (2) and working electrode (1). Working electrode (1) comprises a loop (6) so as to ensure adhesion of polyacrylamide hydrogel, and the counter electrode (2) have relatively large area than the working electrode (1). The invention also relates to a method for producing immobilized micro- encapsulated with minimum loss of enzyme activity. The method comprises steps of spray drying of an emulsion to form encapsulated CAB enzyme powder and immobilizing the encapsulated enzyme powder by hydrogel being formed by bulk polymerization.

Full Text

FIELD OF INVENTION
The present invention relates to an enzyme biosensor device. More particularly, the
invention relates to an improved device for amperometric estimation of carbamate or
organo-phosphorous based pesticides in a given liquid sample by way of improved
immobilization of microencapsulated enzyme thereby enhancing the efficacy of the
device. The invention also relates to a method for producing microencapsulated electrode
for amperometric estimation of pesticides in a given liquid sample.
BACKGROUND AND PRIOR ART
In modern days the rampant use of organophosphorus-based pesticides is creating high
risk of health hazards. Farmers all over the world use organophosphorus-based pesticides
and insecticides extensively.
These neurotoxic compounds are highly toxic because of their inhibitory effect on
cholinesterase enzymes essential for the functioning of the central nervous system in
humans and insects. As acetylcholinesterase (AChE) promotes the hydrolysis of natural
neurotransmitter, acetylcholin (ACh), its inhibition terminates the propagation of the
nerve impulse.
In a recent US geological survey study, widespread presence of trace amounts of these
pesticides was found in surface and ground waters across the US. A similar presence of
the deadly organophosphorus could be expected in the water resources across India.
So, there is a need for development of sensitive methods for the detection of
cholinesterase inhibitors for both environmental monitoring and clinical diagnosis.
Traditionally, pesticides residues could be measured by the methods of liquid
chromatography-mass spectrometry (LC-MS), high performance liquid chromatography
2-

(HPLC), immunoassay e.g. enzyme linked immuno sorbant assay (Elisa) etc. Highly
sensitive HPLC or GC procedures were also developed for the analytical determination of
these pesticides. However, these processes were not very effective, as the amount of
pesticide could not be measured precisely.
Prior art discuss about biosensor, screen-printed biosensor, electrical biosensor, receptor-
based biosensor and spectroscopic methods for detection of chemical toxins.
Most biosensor discussed in prior art consists of enzymes that are immobilized by means
of covalent bonds. Immobilizing the enzymes by means of covalent bond reduces the
enzyme activity and hence efficiency of biosensor.
Immobilization is a process by which the biomaterials are held firmly on the transducer
surface. The process has been widely used by researchers in biosensors. Some examples
are adsorption, covalent bonding, cross-linking etc. The water-in-oil emulsions are
produced with a particular emulsifier such that the aqueous phase is inside a thin layer of
the organic phase (polymer). This was sprayed through a spray drier to form beads of
polymer having the biomaterial inside it.
US6406876 describes a method for detecting hazardous chemicals using biosensor. The
biosensor consists of enzymes that are immobilized on or inside a porous or a non-porous
support by covalent bonds. Covalent bonding of the enzyme with the support reduces
enzyme activity.
US5001048 describes electrical biosensor containing a biological receptor immobilized
and stabilized in a protein film. In case of binding a biological receptor in a protein film
the key mechanism of binding is covalent bonding.
US4324858 describes immobilization of enzyme by impregnation into a porous material.
The porous sheet, which has been used, is difficult to fix on the working electrode. Also
the covalent bonding has been encouraged by using sulphonic, amine groups etc.
3

US5192507 discloses a method of immobilizing and stabilizing an active biological
receptor in a polymeric film and receptor-based biosensor for determining an analyte of
interest in a sample. In case of immobilization of enzyme in a polymeric film by
materials capable of polymerizing and a polymerizing agent may result in covalent
bonding of the enzyme in the polymeric film.
US4411989 describes methods and devices for identifying substances (such as chemical
warfare agents) and residual environmental pollutants (such as pesticides). These utilize
the discovery that spectra of an uninhibited enzyme (e.g., a cholinesterase) can differ
from spectra of the same enzyme, which has been complexed with the agent pollutant.
Analysis by spectroscopic method has lower sensitivity.
None of the prior art discussed above describe bulk polymerization technique for
improved immobilization of microencapsulated enzyme in amperometric estimation of
pesticides for a given liquid sample.
Basic reaction for determination of pesticide :
Enzyme AchE (acetycholine eaterase) hydrolyzes acetylcholine and acetic acid

The choline is oxidized by oxygen catalyzed by the enzyme ChO (choline oxidase) and
H2O2 is produced in the process.

Since H2O2 produced has a very low concentration, it may not be detected by direct
electrochemical route. However good signals can be obtained for a low level (ppb) using
TMB and HRP. The TMB is rapidly oxidized by H2O2 in presence of HRP added to the
system. Due to redox reaction there will be exchange of electrons at the working
electrode.

Thus amperometry can be done for indirect detection of H2O2.
Acetylcholine esterase is partly deactivated by organo-phosphorous (OP) based pesticides
5
due to formation of permanent covalent bond.

Thus there is a need for providing a device for amperometric estimation of pesticides in a
given liquid sample by way of improved immobilization of microencapsulated enzyme
thereby enhancing the efficacy of the device. There is also a need to provide for a method
for producing microencapsulated electrode for amperometric estimation of pesticides in a
given liquid sample.
Thus the present inventors have found that amperometric estimation of carbamate or
organo-phosphorous based pesticides in a given liquid sample can be achieved by way of
improved immobilization of microencapsulated enzyme by bulk polymerization thereby
providing enhancement in overall efficiency of the device.
OBJECTS OF INVENTION
Accordingly, one object of the present invention is to provide a device for amperometric
estimation of pesticides in a given liquid sample by way of improved immobilization of
microencapsulated enzyme.
Another object of the invention is to provide a method for producing microencapsulated
electrode for use in amperometric estimation of pesticides in a given liquid sample.
Another object of the present invention is to provide bulk polymerization.
Yet another object of the invention is to provide a simple, efficient and cost effective
method for amperometric estimation of pesticides.
SUMMARY OF INVENTION
According to one aspect of the present invention there is provided a microencapsulated
enzyme biosensor device for amperometric estimation of carbamate and/or an
organophosphorus based pesticides in a given sample, said device comprising :
6

plurality of electrodes including reference/counter electrode and working electrode,
wherein said working electrode comprising a loop so as to ensure adhesion of
polyacrylamide hydrogel, and
wherein said counter electrode having relatively large area than the working electrode.
Another aspect of the present invention is to provide a method for producing immobilized
micro-encapsulated with minimum loss of enzyme activity, said method comprising steps
of:
spray drying of an emulsion comprising predetermined quantity of acetylcholine esterase
enzyme dissolved in phosphate buffer forming an aqueous phase and organic phase
prepared with cellulose acetate butyrate (CAB) dissolved in dichloromethane (DCM) to
form encapsulated CAB enzyme powder; and
further immobilizing said encapsulated enzyme powder by hydrogel being formed by
bulk polymerization of a mediator selectively comprising acrylamide monomer, N,N
methylene-bis-acrylamide (BIS) and glutaraldehyde and an initiator being selectively
potassium peroxodisulphate.
DETAILED DESCRIPTION OF INVENTION
The present invention is directed to provide a microencapsulated enzyme biosensor for
amperometric estimation of pesticides. The biosensor consists of two electrodes namely
reference/counter electrode made of silver wire and working electrode made of platinum
wire. However, this does not restrict the scope of the present invention. Graphite, glassy
carbon, gold, rhodium etc. can be used as working electrode. But platinum is the best
choice due to its inertness and selectivity. Moreover platinum electrode can operate at a
much lower applied potential compared to that of other electrodes. At higher applied
potential other electrodes also respond to all other electro-active components in the
sample. Silver/silver chloride system or calomel electrode may be used as true reference
7

electrode. Silver may be used as pseudo reference electrode to reduce the size of the three
electrode assembly. Calomel electrode is not preferred due to environmental hazard.
All the wires are coated with some insulation selected from PVC tubing, glass capillary,
epoxy coating etc. and the like. In case of glass capillary or PVC tubing the ends are
sealed by heating or by epoxy. These coated electrodes are set on a polyester matrix using
adhesives. Polymer or glass separators are placed to avoid short circuit between
electrodes. Loops are made at the end of reference and working electrode. The loop at the
terminal of the working electrode ensures adhesion of polyacrylamide hydrogel.
In one preferred embodiment of the invention the terminal of the counter electrode has
been coiled to ensure larger area compared to that of the working electrode.
Preparation of microencapsulated enzyme:
Microencapsulated enzyme is produced by spray drying technique. A mini spray drier
(model no. B191, Buchi, Germany) is used for this purpose with an airflow rate-800 ml/s;
pressure 500 KN/m2 and temperature 45 °C. Measured quantity of acetylcholine esterase
enzyme is dissolved in phosphate buffer pH 7.4 to form the aqueous phase. The organic
phase is prepared with CAB dissolved in dichloromethane (DCM). These two were then
emulsified with SPAN-85. The emulsion then fed into the spray dryer in co current mode.
The encapsulated CAB enzyme powder is separated by the cyclone separator and
collected.
Immobilization of the sensing element on the working electrode:
Microencapsulated AchE (0.03U), ChO (0.03U), HRP (10U) and TMB (0.1 mg) are
immobilized directly on the tip of a Platinum wire (working electrode) by bulk
polymerization of acrylamide monomer, BIS (0 to 10% w/w of amount of monomer,
preferably 4%) and glutaraldehyde (0 to 10% v/w of amount of monomer, preferably 1%)
as crosslinkers, Ferrocene carboxylic acid (0.001 to 0.1% w/w of amount of monomer,
8

preferably 0.01%) as mediator and potassium peroxodisulphate (0.1 to 20% w/w of
amount of monomer, preferably 5%) as initiator.
Bi-functional molecules containing -NH2,-CHO,-COOH as functional groups can be
used as cross linkers. N,N methylene-bis-acrylamide (BIS) is preferred as it forms
irreversible gel. Increase in concentration of BIS makes the gel brittle. On the other hand
glutaraldehyde provides flexibility to the gel. Thus the simultaneous uses of the two cross
linkers helped preparation of a stable and workable gel with proper cross-link density.
Mediators like ferrocene, ferrocene carboxylic acid, potassium ferricyanide etc. can be
used. In the present invention ferrocene carboxylic acid is preferred to ferrocene due to
its solubility in water. Potassium ferricyanide is not preferred due to its inhibitory effect
on polymerization.
Initiators that generate free radicals can be used. Thus initiators like benzoyl peroxide and
ammonium peroxodisulphate can be used. The potassium perdisulphate is preferred as it
ensures faster polymerization at room temperature.
The biosensor device as described above can be used for estimation of any hazardous
chemicals that can deactivate the enzyme Acetylcholine esterase.
Estimation of pesticide: The microencapsulated polymer electrode was dipped in liquid
sample containing pesticide for 15 minutes. It was then washed with buffer and dried in
air. The treated microencapsulated polymer electrode and silver electrode were partly
dipped into phosphate buffer containing 3mM KCL, pH 7.4 and amperometry was done
at -0.25V. The microencapsulated polymer electrode acts as a working electrode and the
silver electrode was used as pseudo reference electrode and counter electrode. After
equilibrium measured quantity of Acetylcholine chloride solution was added.
Reactions for the estimation of pesticides:
The enzyme Acetylcholine esterase hydrolyzes Acetylcholine to choline and acetic acid.
9

Since H2O2 produced has a very low concentration, it may not be detected by direct
electrochemical route. However, good signals are obtained for a low level (ppb) using
TMB and HRP. The TMB is rapidly oxidized by H2O2 in presence of HRP added to the
system. Due to redox reaction there will be exchange of electrons at the working
electrode.

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES
Figure 1 illustrates a schematic diagram of the micro encapsulated biosensor of the
present invention.
Figure 2 illustrates calibration curve for organophosphorous based pesticide.
10

DETAILED DESCRIPTION OF ACCOMPANYING FIGURES
As illustrated in figure 1 the biosensor comprise working platinum electrode (1) which is
placed parallel to reference/counter silver electrode (2) The working electrode (1) as well
as the reference electrode (2) are coated with insulation coating (3). Since the biosensor is
constructed as described above, there can be obtained a biosensor in which the respective
electrodes are surely separated. This separator (4) is placed to avoid short circuit between
electrodes (1) and (2). Two electrodes (1) and (2) are set on a polyester matrix (5). Loop
(6) is made at the end of working electrode (1). Loop (6) is such that it ensures adhesion
of microencapsulated enzyme acrylamide polymer hydrogel (7).
In figure 2 calibration curve for organophosphorous based pesticide is illustrated. With
increase in concentration of the pesticide there will be more deactivation of the enzyme
acetylcholine esterase. Thus at a specified time interval less choline will be produced by
oxidation of the substrate acetylcholine chloride due to the catalytic effect of the
deactivated enzyme AchE.
The amount of hydrogen peroxide produced by the action of ChO is directly proportional
to the amount of choline produced by the earlier reaction. Again the amperometric
response of TMB is proportional to the concentration of hydrogen peroxide.
The graph shows that there is a linear decrease in rate of change in current with increase
in concentration of pesticide. To check whether the sensors were able to give
reproducible results, each experiment was repeated thrice. The error bars based on
standard deviation value indicate that the responses are clustered closely around the mean
i.e., the responses are reproducible.
11

WE CLAIM
1. A microencapsulated enzyme biosensor device for amperometric estimation of
carbamate and/or an organophosphorus based pesticides in a given sample, said
device comprising:
plurality of electrodes including reference/counter electrode and working
electrode,
wherein said working electrode comprising a loop so as to ensure adhesion of
polyacrylamide hydrogel, and wherein said counter electrode having relatively
large area than the working electrode.
2. Device as claimed in claim 1 wherein the counter electrode includes a silver
electrode adapted to be used as pseudo reference electrode.
3. Device as claimed in claim 2 wherein the counter electrode comprises insulation
coating selected from PVC tubing, glass capillary, epoxy coating and the like.
4. Device as claimed in claim 1 wherein the working electrode is a platinum
electrode.
5. Device as claimed in claim 4 wherein the platinum electrode comprises insulation
coating selected from PVC tubing, glass capillary, epoxy coating and the like
6. Device as claimed in any preceding claims further comprising polymer or glass
separator.
7. A method for producing immobilized micro-encapsulated with minimum loss of
enzyme activity, said method comprising steps of:
12

spray drying of an emulsion comprising predetermined quantity of acetylcholine
esterase enzyme dissolved in phosphate buffer forming an aqueous phase and
organic phase prepared with cellulose acetate butyrate (CAB) dissolved in
dichloromethane (DCM) to form encapsulated CAB enzyme powder; and
further immobilizing said encapsulated enzyme powder by hydrogel being formed
by bulk polymerization of a mediator selectively comprising acrylamide
monomer, N,N methylene-bis-acrylamide (BIS) and glutaraldehyde and an
initiator being selectively potassium peroxodisulphate.
I3
8. Method as claimed in claim 7 wherein the phosphate buffer has a pH of 7.4.
9. Method as claimed in claim 7 wherein N,N methylene-bis-acrylamide (BIS) is
present in the range of 0 to 10% w/w, preferably 4% of the amount of monomer.
10. Method as claimed in claim 7 wherein glutaraldehyde is present in the range of 0
to 10% v/w, preferably 1% of the amount of monomer.
11. Method as claimed in claim 7 wherein ferrocene carboxylic acid is further present
in the range of 0.001 to 0.1% w/w, preferably 0.01% of amount of monomer.
12. Method as claimed in claim 7 wherein potassium peroxodisulphate is present in
the range of 0.1 to 20% w/w, preferably 5% of amount of monomer.

The present invention relates to a microencapsulated enzyme biosensor device comprising
plurality of electrodes including reference/counter electrode (2) and working electrode (1).
Working electrode (1) comprises a loop (6) so as to ensure adhesion of polyacrylamide
hydrogel, and the counter electrode (2) have relatively large area than the working
electrode (1). The invention also relates to a method for producing immobilized micro-
encapsulated with minimum loss of enzyme activity. The method comprises steps of spray
drying of an emulsion to form encapsulated CAB enzyme powder and immobilizing the
encapsulated enzyme powder by hydrogel being formed by bulk polymerization.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=n8+DMZoobwjgnWFo/8EW6w==&loc=wDBSZCsAt7zoiVrqcFJsRw==

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Patent Number

272938

Indian Patent Application Number

903/KOL/2007

PG Journal Number

19/2016

Publication Date

06-May-2016

Grant Date

04-May-2016

Date of Filing

21-Jun-2007

Name of Patentee

UNIVERSITY OF CALCUTTA

Applicant Address

SENATE HOUSE, 87/1 COLLEGE STREET, KOLKATA - 700 073

Inventors:

#	Inventor's Name	Inventor's Address
1	BHATTACHARYAY, DIPANKAR	DEPARTMENT OF POLYMER SCIENCE AND TECHNOLOGY, UNIVERSITY OF CALCUTTA, 92 APC ROAD, KOLKATA-700009
2	SARKAR, PRIYABRATA	DEPARTMENT OF POLYMER SCIENCE AND TECHNOLOGY, UNIVERSITY OF CALCUTTA, 92 APC ROAD, KOLKATA-700009
3	GHOSH, DIPANKAR	DEPARTMENT OF POLYMER SCIENCE AND TECHNOLOGY, UNIVERSITY OF CALCUTTA, 92 APC ROAD, KOLKATA-700009

PCT International Classification Number

C12Q1/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA