Indian Patents. 251192:BIO-SENSOR FOR DETECTING ALLERGENS

Title of Invention	BIO-SENSOR FOR DETECTING ALLERGENS
Abstract	There is proposed a bio-sensor screen printed electrode for testing the average levels of dusts / allergens in environment comprising (i) a screen-printed electrode made of a working electrode made of 5% rhodinised carbon, (ii) a reference electrode of 15% Ag/AgCI (iii) and a counter electrode made of carbon formed on a polyester sheet.

Full Text	New patent Filing for Health and Hygiene Sensor in the name of University of Kolkata. (CASE-1) INTRODUCTION TO THE FILED OF INVENTION This invention relates to a very easy to use, handy and disposable electrode for the measurement of over all level of dust/ allergens in the environment. BACKGROUND OF THE INVESTIGATION OF THE INVENTION. There has been a great interest for last two decades on measurement of allergen and dust level in our environment. The fact that allergic individuals have reactions that range from almost unnoticeable to life threatening on exposure to substances is both fascinating and intimidating. Though skin testing has a high degree of sensitivity and specificity for determining antigens that cause allergic disease, positive skin tests do not necessarily indicate that a specific allergen causes symptoms specific for a certain organ. It is therefore necessary that an individual must know the level of allergens in the environment. Allergen source materials are heterogeneous mixtures of proteins, glycoproteins, carbohydrates and other substances that are not allergenic. A simplified procedure of measuring the allergen level is determination of protein content of the sample. We have tried to measure protein in various allergens and real samples from house, Office dusts and also airborne particles (pollen, mite feces, animal hair/dander, moulds) by sucking air for in a controlled way and collecting the deposit and testing it by biosensors. Environmental specimens (dust) from indoor home, school, and work place environments can be evaluated for the content of aeroallergens produced by dust mite, cat, dog, cockroach, and moulds as a means of determining exposure risk and facilitating avoidance therapy. OBJECTS OF THE INVENTION It is a primary object of the invention to propose a screen-printed electrode having counter electrode, a working electrode, a working electrode and a reference electrode. It is another object to propose such an electrode which a screen printed electrode on a substrate of polyester sheet having carbon counter electrode, 5% rhodinised carbon working electrode and Ag/AgCI reference electrode. It is another object to propose a method for the manufacture of screen-printed electrode which can be carried out in house. It is a further object to propose a systematic method for the estimation of protein contact of a desired sample. BRIEF STATEMENT OF THE INVENTION According to this invention there is provided a bio-sensor screen printed electrode for testing the average levels of dusts / allergens in environment comprising (i) a screen-printed electrode made of a working electrode made of 5% rhodinised carbon, (ii) a reference electrode of 15% Ag/AgCI and, (iii) a counter electrode made of carbon formed on a polyester sheet. The electrode is a disposable electrode and is a flow cell electrode. DETAILED STATEMENT OF THE INVENTION WITH REFERENCE TO THE ACCOMPANYING DRAWINGS. The electrochemical activity was measured on a dual potential electrode and also on a bi-enzyme electrode for protein using a dual potential electrode. In a bienzyme electrode, amino acid oxidase was immobilized on the electrode surface and protease was immobilized on ABC membrane which was kept on the working electrode surface in close contact. The analyte either allergen or dust in an electrolyte was added on to the ABC membrane. The protein present in the sample was hydrolised by the protease to form amino acid that in turn produced hydrogen peroxide in presence of amino acid oxidase. This hydrogen peroxide was easily detected amperometrically on the electrode at a potential of 0.35V. In case of a dual potential electrode, the electrode was subjected to a high potential (1.5 V) for electrolysis of KBR to K+ and Br" and the protein content in the sample reacted with the bromine at a lower voltage (0.5V) giving rise to a positive response. The invention is explained further with reference to the accompanying drawings, wherein, Figure 1 shows the schematic diagram of the screen-printed electrode assembly. Figure 2 gives the response of various allergens with a dual potential electrode. The allergens gave very high response. Similarly, figure 3 gave he response in the same order for the allergens of figure 2. Finally figure 4 gives the response with a dual potential electrode for various amount of house dust. The knowledge from these tests can allow health care workers to predict who is at risk for future difficulties related to allergen exposures. The allergen burden has thus become an analytical measurement provided by a simple test at site and will enhance the awareness. Disposable sensor: Three electrode devices were mass manufactured in-house by a multistage screen-printing process using a DEK 248 machine Devices were printed on to 250 urn thick polyester sheet The sensor consisted of a working electrode made of 5% rhodinised carbon, a reference electrode of 15% Ag/AgCI and a counter electrode made of carbon. The schematic of the sensor is shown in Figure 1. Flow cell: The cell was made of two polymethylmethacrylate blocks having a grooved design to fit the screen-printed electrode manufactured as described above. The analyte solution was passed through the cell covering the three electrodes by a peristaltic pump at a measured rate periodically. Methodology Two different techniques were applied to measure the protein content of the sample. First one used a dual potential electrode where an electrolyte, 0.1M KBr solution in buffer, was first electrolyzed at a high voltage (1.6 V) and then the Bromine produced was consumed at a lower potential (0.6V) causing release of electrons as follows: 2Br=Br2 + 2e" (1) The experiments were conducted on screen-printed disposable as well as flow cell electrodes. The second one used a bienzyme electrode, where the protein was first hydrolyzed into amino acid by the enzyme protease and then the amino acid was detected by amino acid oxidase. The hydrogen peroxide thus produced was measured at appropriate potential across the working electrode. H^ —? 2H+ +02+2e" (4) A unique approach was used to immobilize one of the two enzymes by micro encapsulation with a polymer, cellulose acetate butyrate (CAB). The advantages were two folds. Firstly a separate membrane such as immunodyne ABC was not required to make a barrier between the two enzymes. Secondly due to micro encapsulation, the enzyme protease was held firmly inside the microcapsules and these could be repeatedly used without loss of activity. A two-step emulsification procedure was adopted to produce the microcapsules. The first emulsion was produced was produced by 2 ml double distilled cellulose acetate butyrate in dichloromethane) using a sonicator (Sonics and Materials Inc, USA, VCX 600) with span -85 (200 ul) to make w/o emulsion. This emulsion was added slowly sprayed to external aqueous (15 ml) phase having 0.5 % w/v poly (vinyl alcohol) and 300 ul Tween 80 to yield a w/o/w multiple emulsion. The multiple emulsion was stirred to drive away DCM. The microcapsules thus obtained were separated in a Hettich (Universal 32) centrifuge at 8000 rpm for 30 minutes, washed and allowed to dry at 4° C for 24 hours. These were mounted on the working electrode of the transducer which was already coated with L-and D-amino acid oxidase enzyme. The mechanism of this electrode involves transport of analyte into the microcapsules thereby generating amino acid which diffuses out of the capsules and reacts with the amino acid oxidase enzymes to produce hydrogen peroxide. INSTRUMENTATION The test procedures were controlled using an AutoLab Electrochemical; Analyzer (u AutoLab - Type II) with GPES software. The response of an individual electrode was obtained by deducting the base line current, from the response at a particular set of conditions (e.g. enzyme loading concentration of substrate, time of measurement). The average value of this difference was plotted against concentrations or strengths. ALLERGEN EXTRACTS Allergen extracts were prepared from a wide variety of source materials including pollens, fungi, arthropods, foods and dusts. Allergens: All allergens were received from Biopol Laboratory Inc., USA. The allergens were 1. Cynodon Dactycon (Bermuda grass pollen), 2. Poa Pratensis (Kentucky bluegrass pollen), 3. Fragaria Ananassa (Strawberry), 4. Nicotiana Tabacum (green tobacco leaf), 5. Ustilago Maydis (Corn smut), 6. Dermatophagolies Farinae Faces (Dust mite faces), 7. Zea Mays (Corn pollen), 8. Medicago Sativa (Alfalfa pollen), 9. Cockroach Detrius, 10. Saccharomyces Cerevisia (Brewer yeast) 11. Penicillium Notatum, 12. Felis Sylvestris (Cat hair), 13. Gallus Gallus (chicken feathers), 14. Melop Sittacus Undulatus (Parakeet feathers), 15. Ustilago Tritici (Loose wheat smut), 16. Ambrosia Trifida (Giant ragweed pollen), 17. Lolium Perenne (Perennial ryegrass pollen), 18. Rumex Crispus (Carly dock pollen), Dust was collected from Office, home and laboratory. The enzymatic activities of different allergens and dusts were measured. 50 mg of the solid samples were taken in 1 ml phosphate buffer. 5 Fsymbol ml of each extraction fluids of allergens was added on different electrodes containing 50 ul phosphate-buffer with KCL (0.1M) using o-ring. The protein content of each allergen was estimated by I) a dual potential method, where the voltage was applied in two steps i.e. 1. 6V and 0.6V (duration 120 sec each), ii) bienzyme electrode, where the electrode was poised at +0.35 V across the working electrode. RESULTS AND DISCUSSIONS. Figure 2 shows transient electrochemical activity of samples on the bare electrode i.e. not modified with enzyme when a potential of 0.35V was applied across the working electrode, the strength of the allergen was kept at from 0.25 mg in 55 ul (extract). Cockroach detritus and green tobacco leaf showed a high response. This was due to the presence of electrochemically active constituents present in these allergens. All other allergens tested gave insignificant response on a bare electrode. Figure 3 shows the response of some allergens (Cockroach Detrius, Alfalfa Pollen (Medicago Sativa), Corn Pollen (Zea Mays), Perennial Ryegrass Pollen (Lolium Perenne), Corn smut (Ustilago Maydis), Parakeet feathers (Melop Sittacus Undulatus), Cat hair (Felis Sylvestris) on a dual potential electrode using KBr as a reaction medium for protein. Almost all gave quite good response. The order remains the same when analyzed by-enzyme system using microencapsulated protease (Figure 4). We have also analyzed the samples in a flow cell using dual potential method and the results are depicted in Figure 5. The amplitudes are different but the order remains the same. The difference of amplitude is due to the variation of surface area of disposable electrode and a flow cell electrode. Figure 6 depicts response from house dust samples of various quantities on a dual potential electrode. Thus this would give a very good indication of the dust level in the house and the device may be used as an indicator of the dust in a living environment. WE CLAIM 1)A bio-sensor screen printed electrode for testing the average levels of dusts / allergens in environment comprising (i) a screen-printed electrode made of a working electrode made of 5% rhodinised carbon, (ii) a reference electrode of 15% Ag/AgCI, (iii) and a counter electrode made of carbon formed on a polyester sheet. 2) A bio-sensor screen printed electrode as claimed in Claim 1 wherein the electrode is a disposable electrode. 3) A bio-sensor screen printed electrode as claimed in Claim 2 wherein the electrode is a flow cell electrode. 4) A bio sensor screen printed electrode substantially as herein described with reference to the accompanying drawings. There is proposed a bio-sensor screen printed electrode for testing the average levels of dusts / allergens in environment comprising (i) a screen-printed electrode made of a working electrode made of 5% rhodinised carbon, (ii) a reference electrode of 15% Ag/AgCI (iii) and a counter electrode made of carbon formed on a polyester sheet.

Full Text

New patent Filing for Health and Hygiene Sensor in the
name of University of Kolkata. (CASE-1)
INTRODUCTION TO THE FILED OF INVENTION
This invention relates to a very easy to use, handy and disposable electrode for the measurement of
over all level of dust/ allergens in the environment.
BACKGROUND OF THE INVESTIGATION OF THE INVENTION.
There has been a great interest for last two decades on measurement of allergen and dust level in our
environment. The fact that allergic individuals have reactions that range from almost unnoticeable to
life threatening on exposure to substances is both fascinating and intimidating. Though skin testing
has a high degree of sensitivity and specificity for determining antigens that cause allergic disease,
positive skin tests do not necessarily indicate that a specific allergen causes symptoms specific for a
certain organ. It is therefore necessary that an individual must know the level of allergens in the
environment.
Allergen source materials are heterogeneous mixtures of proteins, glycoproteins, carbohydrates and
other substances that are not allergenic. A simplified procedure of measuring the allergen level is
determination of protein content of the sample. We have tried to measure protein in various allergens
and real samples from house, Office dusts and also airborne particles (pollen, mite feces, animal
hair/dander, moulds) by sucking air for in a controlled way and collecting the deposit and testing it by
biosensors.
Environmental specimens (dust) from indoor home, school, and work place environments can be
evaluated for the content of aeroallergens produced by dust mite, cat, dog, cockroach, and moulds as
a means of determining exposure risk and facilitating avoidance therapy.
OBJECTS OF THE INVENTION
It is a primary object of the invention to propose a screen-printed electrode having counter electrode, a
working electrode, a working electrode and a reference electrode.
It is another object to propose such an electrode which a screen printed electrode on a substrate of
polyester sheet having carbon counter electrode, 5% rhodinised carbon working electrode and
Ag/AgCI reference electrode.
It is another object to propose a method for the manufacture of screen-printed electrode which can be
carried out in house.
It is a further object to propose a systematic method for the estimation of protein contact of a desired
sample.
BRIEF STATEMENT OF THE INVENTION
According to this invention there is provided a bio-sensor screen printed electrode for testing the
average levels of dusts / allergens in environment comprising
(i) a screen-printed electrode made of a working electrode made of 5% rhodinised carbon,
(ii) a reference electrode of 15% Ag/AgCI and,
(iii) a counter electrode made of carbon formed on a polyester sheet.
The electrode is a disposable electrode and is a flow cell electrode.
DETAILED STATEMENT OF THE INVENTION WITH REFERENCE TO THE ACCOMPANYING
DRAWINGS.
The electrochemical activity was measured on a dual potential electrode and also on a bi-enzyme
electrode for protein using a dual potential electrode.
In a bienzyme electrode, amino acid oxidase was immobilized on the electrode surface and protease
was immobilized on ABC membrane which was kept on the working electrode surface in close contact.
The analyte either allergen or dust in an electrolyte was added on to the ABC membrane. The protein
present in the sample was hydrolised by the protease to form amino acid that in turn produced
hydrogen peroxide in presence of amino acid oxidase. This hydrogen peroxide was easily detected
amperometrically on the electrode at a potential of 0.35V.
In case of a dual potential electrode, the electrode was subjected to a high potential (1.5 V) for
electrolysis of KBR to K+ and Br" and the protein content in the sample reacted with the bromine at a
lower voltage (0.5V) giving rise to a positive response.
The invention is explained further with reference to the accompanying drawings, wherein,
Figure 1 shows the schematic diagram of the screen-printed electrode assembly.
Figure 2 gives the response of various allergens with a dual potential electrode.
The allergens gave very high response.
Similarly, figure 3 gave he response in the same order for the allergens of figure 2.
Finally figure 4 gives the response with a dual potential electrode for various amount of house dust.
The knowledge from these tests can allow health care workers to predict who is at risk for future
difficulties related to allergen exposures. The allergen burden has thus become an analytical
measurement provided by a simple test at site and will enhance the awareness.
Disposable sensor: Three electrode devices were mass manufactured in-house by a multistage
screen-printing process using a DEK 248 machine Devices were printed on to 250 urn thick polyester
sheet The sensor consisted of a working electrode made of 5% rhodinised carbon, a reference
electrode of 15% Ag/AgCI and a counter electrode made of carbon. The schematic of the sensor is
shown in Figure 1.
Flow cell: The cell was made of two polymethylmethacrylate blocks having a grooved design to fit the
screen-printed electrode manufactured as described above. The analyte solution was passed through
the cell covering the three electrodes by a peristaltic pump at a measured rate periodically.
Methodology
Two different techniques were applied to measure the protein content of the sample. First one used a
dual potential electrode where an electrolyte, 0.1M KBr solution in buffer, was first electrolyzed at a
high voltage (1.6 V) and then the Bromine produced was consumed at a lower potential (0.6V) causing
release of electrons as follows:
2Br=Br2 + 2e" (1)
The experiments were conducted on screen-printed disposable as well as flow cell electrodes. The
second one used a bienzyme electrode, where the protein was first hydrolyzed into amino acid by the
enzyme protease and then the amino acid was detected by amino acid oxidase.

The hydrogen peroxide thus produced was measured at appropriate potential across the working
electrode.
H^ —? 2H+ +02+2e" (4)
A unique approach was used to immobilize one of the two enzymes by micro encapsulation with a
polymer, cellulose acetate butyrate (CAB). The advantages were two folds. Firstly a separate
membrane such as immunodyne ABC was not required to make a barrier between the two enzymes.
Secondly due to micro encapsulation, the enzyme protease was held firmly inside the microcapsules
and these could be repeatedly used without loss of activity. A two-step emulsification procedure was
adopted to produce the microcapsules. The first emulsion was produced was produced by 2 ml double
distilled cellulose acetate butyrate in dichloromethane) using a sonicator (Sonics and Materials Inc,
USA, VCX 600) with span -85 (200 ul) to make w/o emulsion. This emulsion was added slowly
sprayed to external aqueous (15 ml) phase having 0.5 % w/v poly (vinyl alcohol) and 300 ul Tween 80
to yield a w/o/w multiple emulsion. The multiple emulsion was stirred to drive away DCM. The
microcapsules thus obtained were separated in a Hettich (Universal 32) centrifuge at 8000 rpm for 30
minutes, washed and allowed to dry at 4° C for 24 hours. These were mounted on the working
electrode of the transducer which was already coated with L-and D-amino acid oxidase enzyme. The
mechanism of this electrode involves transport of analyte into the microcapsules thereby generating
amino acid which diffuses out of the capsules and reacts with the amino acid oxidase enzymes to
produce hydrogen peroxide.
INSTRUMENTATION
The test procedures were controlled using an AutoLab Electrochemical; Analyzer (u AutoLab - Type
II) with GPES software.
The response of an individual electrode was obtained by deducting the base line current, from the
response at a particular set of conditions (e.g. enzyme loading concentration of substrate, time of
measurement). The average value of this difference was plotted against concentrations or strengths.
ALLERGEN EXTRACTS
Allergen extracts were prepared from a wide variety of source materials including pollens, fungi,
arthropods, foods and dusts. Allergens: All allergens were received from Biopol Laboratory Inc., USA.
The allergens were 1. Cynodon Dactycon (Bermuda grass pollen), 2. Poa Pratensis (Kentucky
bluegrass pollen), 3. Fragaria Ananassa (Strawberry), 4. Nicotiana Tabacum (green tobacco leaf),
5. Ustilago Maydis (Corn smut), 6. Dermatophagolies Farinae Faces (Dust mite faces), 7. Zea Mays
(Corn pollen), 8. Medicago Sativa (Alfalfa pollen), 9. Cockroach Detrius, 10. Saccharomyces Cerevisia
(Brewer yeast) 11. Penicillium Notatum, 12. Felis Sylvestris (Cat hair), 13. Gallus Gallus (chicken
feathers), 14. Melop Sittacus Undulatus (Parakeet feathers), 15. Ustilago Tritici (Loose wheat smut),
16. Ambrosia Trifida (Giant ragweed pollen), 17. Lolium Perenne (Perennial ryegrass pollen),
18. Rumex Crispus (Carly dock pollen), Dust was collected from Office, home and laboratory.
The enzymatic activities of different allergens and dusts were measured. 50 mg of the solid samples
were taken in 1 ml phosphate buffer. 5 Fsymbol ml of each extraction fluids of allergens was added on different
electrodes containing 50 ul phosphate-buffer with KCL (0.1M) using o-ring.
The protein content of each allergen was estimated by I) a dual potential method, where the voltage
was applied in two steps i.e. 1. 6V and 0.6V (duration 120 sec each), ii) bienzyme electrode, where the
electrode was poised at +0.35 V across the working electrode.
RESULTS AND DISCUSSIONS.
Figure 2 shows transient electrochemical activity of samples on the bare electrode i.e. not modified
with enzyme when a potential of 0.35V was applied across the working electrode, the strength of the
allergen was kept at from 0.25 mg in 55 ul (extract). Cockroach detritus and green tobacco leaf
showed a high response. This was due to the presence of electrochemically active constituents
present in these allergens. All other allergens tested gave insignificant response on a bare electrode.
Figure 3 shows the response of some allergens (Cockroach Detrius, Alfalfa Pollen (Medicago Sativa),
Corn Pollen (Zea Mays), Perennial Ryegrass Pollen (Lolium Perenne), Corn smut (Ustilago Maydis),
Parakeet feathers (Melop Sittacus Undulatus), Cat hair (Felis Sylvestris) on a dual potential electrode
using KBr as a reaction medium for protein. Almost all gave quite good response. The order remains
the same when analyzed by-enzyme system using microencapsulated protease (Figure 4). We have
also analyzed the samples in a flow cell using dual potential method and the results are depicted in
Figure 5. The amplitudes are different but the order remains the same. The difference of amplitude is
due to the variation of surface area of disposable electrode and a flow cell electrode. Figure 6 depicts
response from house dust samples of various quantities on a dual potential electrode. Thus this would
give a very good indication of the dust level in the house and the device may be used as an indicator
of the dust in a living environment.
WE CLAIM
1)A bio-sensor screen printed electrode for testing the average levels of dusts / allergens in
environment comprising
(i) a screen-printed electrode made of a working electrode made of 5% rhodinised carbon,
(ii) a reference electrode of 15% Ag/AgCI,
(iii) and a counter electrode made of carbon formed on a polyester sheet.
2) A bio-sensor screen printed electrode as claimed in Claim 1 wherein the electrode is a disposable
electrode.
3) A bio-sensor screen printed electrode as claimed in Claim 2 wherein the electrode is a flow cell
electrode.
4) A bio sensor screen printed electrode substantially as herein described with reference to the
accompanying drawings.

There is proposed a bio-sensor screen printed electrode for testing the average levels of dusts /
allergens in environment comprising
(i) a screen-printed electrode made of a working electrode made of 5% rhodinised carbon,
(ii) a reference electrode of 15% Ag/AgCI
(iii) and a counter electrode made of carbon formed on a polyester sheet.

Documents:

121-KOL-2003-(06-09-2011)-CORRESPONDENCE.pdf

121-KOL-2003-(11-11-2011)-ABSTRACT.pdf

121-KOL-2003-(11-11-2011)-AMANDED CLAIMS.pdf

121-KOL-2003-(11-11-2011)-CORRESPONDENCE.pdf

121-KOL-2003-(11-11-2011)-FORM 1.pdf

121-KOL-2003-(11-11-2011)-FORM 2.pdf

121-KOL-2003-(11-11-2011)-PA.pdf

121-KOL-2003-(20-10-2011)-CORRESPONDENCE.pdf

121-KOL-2003-ABSTRACT-1.1.pdf

121-kol-2003-abstract.pdf

121-KOL-2003-AMANDED CLAIMS.pdf

121-kol-2003-claims.pdf

121-KOL-2003-CORRESPONDENCE 1.1.pdf

121-KOL-2003-CORRESPONDENCE-1.2.pdf

121-kol-2003-correspondence.pdf

121-kol-2003-description (complete).pdf

121-KOL-2003-DRAWINGS-1.1.pdf

121-kol-2003-drawings.pdf

121-KOL-2003-EXAMINATION REPORT REPLY RECIEVED.pdf

121-KOL-2003-FORM 1-1.1.pdf

121-kol-2003-form 1.pdf

121-kol-2003-form 13.pdf

121-kol-2003-form 18.pdf

121-KOL-2003-FORM 2-1.1.pdf

121-kol-2003-form 2.pdf

121-KOL-2003-FORM 3-1.1.pdf

121-kol-2003-form 3.pdf

121-KOL-2003-OTHERS.pdf

121-kol-2003-pa.pdf

121-kol-2003-specification.pdf

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Patent Number

251192

Indian Patent Application Number

121/KOL/2003

PG Journal Number

09/2012

Publication Date

02-Mar-2012

Grant Date

29-Feb-2012

Date of Filing

27-Feb-2003

Name of Patentee

CALCUTTA UNIVERSITY

Applicant Address

92, ACHARYA PRAFFULA CHANDRA ROAD, CALCUTTA

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. PRIYOBRATA SARKAR	92, ACHARYA PRAFFULA CHANDRA ROAD, CALCUTTA - 700 009

PCT International Classification Number

G01N 27/327

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA