DESC:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
The Patents Rules, 2003
COMPLETE SPECIFICATION
(Section 10 and Rule 13)
TITLE
A Method to Detect Concentration of Residual Pesticides
APPLICANT
(a) Bharuwa Agri Science Pvt. Limited.
(b) An Indian Company,
(c) Divya Yog Mandir, Room No. 07,
Dadubagh, Kankhal, Haridwar – 249408,
Uttarakhand, India
PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in
which it is to be performed.
2
FIELD OF THE INVENTION
This invention relates to a method to detect concentration of the residual
pesticides. Particularly this invention relates to a method to detect concentration
of the residual pesticides available in vegetables, fruits, eatables, and drinkable
materials. 5
BACKGROUND OF THE INVENTION
It is noticed that a lot of pesticides are used to grow different crops including
vegetables and fruit crops just to safeguard the crips from different kinds of pests.
The pesticides used in the crops normally percolate in the fruits and vegetables.
Sometimes presence of the pesticides in fruits and vegetables is more than the 10
allowable limits. It is also observed that many diseases are caused in human
beings and even animals due to the excess amount of pesticides present in
vegetables and fruits. There are methods and systems known in the prior art to
detect pesticides available in fruits and vegetables.
Chinese patent (CN105044101A) discloses a quick test card for pesticide 15
residues, comprising: a fixed film, a sample piece, a substrate piece, and a test
strip; wherein the sample piece, the substrate piece, and the test strip are all paper
materials, which are superposed from top to bottom; The upper and lower sides
are closed by a fixed film; a sample hole is provided on the fixed film
corresponding to the position of the sample piece for dropping the sample 20
solution; the sample piece is adsorbed and fixed with acetylcholinesterase, the
substrate The sheet is adsorbed and immobilized with thioacetylcholine iodide,
and the detection strip is provided with a ribbon at equal intervals.
US patent (US9417206B2) discloses an apparatus for residual pesticide
detection, comprising: a disposable electrode strip body having an insulating 25
substrate, the disposable electrode strip body being divided into a first strip portion
and a second strip portion, the first strip portion being with an electrode pattern
placed on the insulating substrate, the second strip portion being without an
3
electrode pattern placed on the insulating substrate, a splitting line being located
between the first strip portion and the second strip portion and being between the
insulating substrate, the splitting line being split to separate the first strip portion
and the second strip portion from each other and separate the insulating substrate
apart, wherein the first strip portion is provided with a working electrode area 5
having a predetermined amount of a first reactant thereon, and the second strip
portion having a predetermined amount of a second reactant thereon;
a mixing container for containing a predetermined volume of sample liquid to be
tested and for containing the second strip portion, wherein the second reactant of
the second strip portion is dissolved absolutely in the sample liquid to form mixed 10
aqueous solution; and
an electrical signal analyzer, connected to the first strip portion, for detecting an
electrical current signal to obtain normality of pesticide residue in the sample liquid
as compared to a predetermined pesticide, wherein the electrical current signal is
detected by: introducing the mixed aqueous solution into the working electrode 15
area via a micro-channel of the first strip portion to react with the first reactant for
a time period, and applying a voltage to the first strip portion.
The publication “Rapid detection of pesticide in milk, cereal and cereal based
food and fruit juices using paper strip-based sensor” is aimed to validate
paper strip sensors for the detection of pesticide residues in milk, cereal-based 20
food, and fruit juices in comparison with GC–MS/MS under field conditions. The
detection limit of pesticide using rapid paper strip sensor for organophosphate,
carbamate, organochlorine, fungicide, and herbicide group ranges from 1 to 10,
1–50, 250–500, 1–50, and 1 ppb, respectively in milk and milk product, cereal?based food and fruit juices. Among 125 samples of milk samples collected from 25
the market 33 milk samples comprising 31 raw milk and 2 pasteurized milk found
positive for pesticide using the strip-based sensor. In cereal based food and fruit
juice samples, 6 cereal flours and 4 fruit juices were found positive for pesticide
4
residues. The pesticide positive samples were further evaluated quantitatively
using GC–MS/MS wherein 7 samples comprised of raw milk, pasteurized milk,
rice flour, wheat flour, maize flour, apple juice, and pomegranate juice have shown
the presence of chlorpyrifos, chlorpyrifos-methyl, a-endosulfan, ß-endosulfan
DDD and DDT at trace level as well as at above MRL level. It is envisaged that 5
the developed paper strip sensor can be a potential tool in the rapid and cost?effective screening of a large number of food samples for pesticide residues.
The publication “Optical Screening Methods for Pesticide Residue Detection
in Food Matrices: Advances and Emerging Analytical Trends” reviews the
available screening methods for pesticide residues on the basis of optical 10
detection during the period 2016–2020. Optical methods are commonly
miniaturized analytical platforms introducing the point-of-care (POC) era in the
field. Various optical detection principles have been utilized, namely, colorimetry,
fluorescence (FL), surface plasmon resonance (SPR), and surface enhanced
Raman spectroscopy (SERS). Nanomaterials can significantly enhance optical 15
detection performance and handheld platforms, for example, handheld SERS
devices can revolutionize testing. The hyphenation of optical assays to
smartphones is also underlined as it enables unprecedented features such as
one-click results using smartphone apps or online result communication. All in all,
despite being in an early stage facing several challenges, i.e., long sample 20
preparation protocols or interphone variation results, such POC diagnostics pave
a new road into the food safety field in which analysis cost will be reduced and a
more intensive testing will be achieved.
There are disadvantages associated with conventional methods. One of the
disadvantages is that conventional methods are not user friendly and trained 25
people are needed to perform the tests.
Another disadvantage associated with conventional methods is that conventional
methods do not offer high sensitivity of detecting pesticides.
5
Yet another disadvantage associated with conventional methods is that
conventional methods are lengthy and the users have to wait for a long time.
Therefore, there is a need to invent a method to detect the presence of pesticides
in the fruits and vegetables.
OBJECTS OF THE INVENTION 5
Therefore, an object of the present invention is to provide a method to detect
concentration of the residual pesticides, which obviates the disadvantages
associated with conventional systems and methods.
Another object of the present invention is to provide a method to detect
concentration of the residual pesticides, which offers high sensitivity in detecting 10
pesticide residues. The colour change from blue to green provides a clear visual
indicating that the concentration of the pesticide in the sample is below the
specified threshold or equal / above to specified threshold.
Yet another object of the present invention is to provide a method to detect
concentration of the residual pesticides, which is simple and can be performed 15
easily. The steps involved in sample preparation are easy and therefore make the
process user-friendly.
Still, another object of the present invention is to provide a method to detect
concentration of the residual pesticides, which is quick and the colour change is
observed in less time with clear indication about the presence of the pesticide. 20
A further object of the present invention is to provide a method to detect
concentration of the residual pesticide which is optimized and calibrated to
correspond to specific pesticide concentrations so as ensure accurate and reliable
results for the precise presence of the pesticide concentration in the sample.
SUMMARY OF THE INVENTION 25
6
According to this invention, there is provide a method to detect concentration of
residual pesticides available in fruits and vegetables comprising preparing a
pesticide sample of fruit or vegetable and taking 80 - 120 µl of the same into a
tube (A),adding 20 – 40 µl of enzyme solution into the tube (A) and incubating the
same for 5 – 15 minutes at room temperature to obtain enzymatic 5
solution,preparing a solution by mixing homogeneously 400 - 500 µl of Sodium
phosphate buffer, 40 - 60 µl of Acetylcholinethioiodide, and 40 – 60 µl of
Bromothymol Blue in another tube (B),adding the enzymatic solution of the tube
(A) into solution of another tube (B) and incubating the same for 10 – 20 minutes,
observing color change of the solution for initial detection of concentration of 10
residual pesticides available in fruits and vegetables, incubating the above
mixture for another 10 – 20 minutes and observing the color change of the solution
for confirmation of detection of concentration of residual pesticides available in
fruits and vegetables.
Further according to the invention, the pesticide sample of fruits and vegetable is 15
prepared by taking 80 - 120 grams of the desired fruits or vegetables and cutting
the same into small pieces, placing the cut pieces into a container and adding 80
- 120 ml of water into the container, shaking the container intermittently for 8 - 12
minutes to facilitate the transfer of pesticides from the vegetable surface into the
water, allowing the mixture to settle for a few minutes, decanting aqueous layer 20
from the top of the container such that to leave behind the solid fruits or vegetable
material to obtain pesticide sample.
BRIEF DESCRIPTION OF THE DRAWINGS
A method to detect concentration of the residual pesticide, according to a
preferred embodiment of the present invention, is herein described and illustrated 25
in the accompanying drawings, wherein:
Figure 1 (a, & b) – illustrates a tube A and tube B,
Figure 2 – illustrates multiple samples of negative, positive sample of the
experiment 1,
7
Figure 3 – illustrates multiple samples of negative, positive sample of the
experiment 2,
Figure 4 & 5 - illustrates multiple samples of negative, positive sample of the
experiment 3,
Figure 6 &7 – illustrates multiple samples of negative, positive sample of the 5
experiment 4,
Figure 8 – illustrates multiple samples of negative, positive sample of the
experiment 5,
Figure 9 – illustrates multiple samples of negative, positive sample of the
experiment 6. 10
Figure 10 – illustrates multiple samples of negative, positive sample of the
experiment 7.
Figure 11 – illustrates multiple samples of negative, positive sample of the
experiment 8.
Figure 12 – illustrates multiple samples of negative, positive sample of the 15
experiment 9.
DETAILED DESCRIPTION OF THE INVENTION
A method to detect concentration of the residual pesticide is herein described and
illustrated with numerous specific details so as to provide a complete
understanding of the invention. However, these specific details are exemplary 20
details and should not be treated as the limitation to the scope of the invention.
The invention may be performed with slight modifications. Throughout this
specification the word “comprise” or variations such as “comprises or comprising”,
will be understood to imply the inclusions of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusions of any other element, 25
integer or step or group of elements, integers or steps.
8
A method to detect concentration of residual pesticides available in fruits and
vegetables comprises, a pesticide sample of fruit or vegetable is prepared and
taken 80 - 120 µl of the same into a tube (A). 20 – 40 µl of enzyme solution, for
example, 0.01 – 0.03 U/µl of concentration and pH value between 6.8 – 7.2 of the
solution, is added into the tube (A) and incubated the same for 5 – 15 minutes at 5
room temperature to obtain enzymatic solution. A solution is prepared by mixing
homogeneously 400 - 500 µl of Sodium phosphate buffer, 40 - 60 µl of
Acetylcholinethioiodide, and 40 – 60 µl of Bromothymol Blue in another tube (B).
The enzymatic solution of the tube (A) is added into solution of another tube (B)
and incubated the same for 10 – 20 minutes, during the incubation, observing 10
color change of the solution for initial detection of concentration of residual
pesticides available in fruits and vegetables. Further, the above mixture is
incubated for another 10 – 20 minutes and observing the color change of the
solution for confirmation of detection of concentration of residual pesticides
available in fruits and vegetables. 15
The change of color of the solution from blue to green, is indicated that the
pesticides concentration in the sample is below a specified threshold. The
unchanged of color of the solution, is indicated that the pesticides concentration
in the sample is above the specified threshold. The specified threshold value is
depended on the sample of multiple type of fruits and vegetable. 20
The sodium phosphate buffer is used of a concentration 45 - 55 mM sodium
phosphate buffer and pH value between 7.6 – 8.0.
The pesticide sample of fruits and vegetable is prepared by taking 80 - 120 grams
of the desired fruits or vegetables and cut the same into small pieces. The cut
pieces are placed into a container and added 80 - 120 ml of water into the 25
container. The container is intermittently shaked for 8 - 12 minutes to facilitate the
transfer of pesticides from the vegetable surface into the water. The mixture is
allowed to settle for a few minutes, decanted aqueous layer from the top of the
9
container such that to leave behind the solid fruits or vegetable material to obtain
pesticide sample.
EXAMPLE
The pesticide sample of a fruit was prepared by taking 90 grams of a fruit and cut
the same into small pieces. The cut pieces were placed into a container and 5
added 90 ml of water into the container such that to completely deep in the pieces.
The container was intermittently shaked for 11 minutes to facilitate the transfer of
pesticides from the vegetable surface into the water. The mixture was allowed to
settle for a few minutes, decanted aqueous layer from the top of the container
such that to leave behind the solid fruits or vegetable material to obtain pesticide 10
sample.
The pesticide sample of fruit, 110 µl of the sample was taken into a tube (A). 32
µl of enzyme solution of 0.02 U/µl of concentration and pH value 7 of the solution,
was added into the tube (A) and incubated the same for 12 minutes at room
temperature to obtain enzymatic solution. A solution was prepared by mixing 15
homogeneously 460 µl of Sodium phosphate buffer, 50 µl of
Acetylcholinethioiodide, and 45 µl of Bromothymol Blue in another tube (B). The
enzymatic solution of the tube (A) was added into solution of another tube (B) and
incubated the same for 16 minutes, during the incubation, it observed that the
color of the solution is unchanged. Further, the above mixture was incubated for 20
another 18 minutes and it observed that the color change of the solution remain
unchanged. This was show that the pesticides concentration in the sample is
above the specified threshold.
EXAMPLE -2
The pesticide sample of a vegetable was prepared by taking 95 grams of a 25
vegetable and cut the same into small pieces. The cut pieces were placed into a
container and added 100 ml of water into the container such that to completely
10
deep therein. The container was intermittently shaked for 9 minutes to facilitate
the transfer of pesticides from the vegetable surface into the water. The mixture
was allowed to settle for a few minutes, decanted aqueous layer from the top of
the container such that to leave behind the solid fruits or vegetable material to
obtain pesticide sample. 5
The pesticide sample of vegetable, 100 µl of the sample was taken into a tube
(A). 30 µl of enzyme solution of 0.02 U/µl of concentration and pH value 7.1 of the
solution, was added into the tube (A) and incubated the same for 13 minutes at
room temperature to obtain enzymatic solution. A solution was prepared by mixing
homogeneously 450 µl of Sodium phosphate buffer, 50 µl of 10
Acetylcholinethioiodide, and 45 µl of Bromothymol Blue in another tube (B). The
enzymatic solution of the tube (A) was added into solution of another tube (B) and
incubated the same for 16 minutes, during the incubation, it observed that the
color of the solution is unchanged. Further, the above mixture was incubated for
another 19 minutes and it observed that the color change of the solution is 15
changed from blue to green. This was show that the pesticides concentration in
the sample is below the specified threshold.
The present invented method was tested with multiple sample and experiments.
The tested method and experiment result are mentioned as follows.
The objective is to detect a total of 12 pesticides, consisting of 8 organophosphate 20
pesticides: Methyl parathion, Parathion, Monocrotophos, Chlorpyrifos, Phorate,
Profenfos, Quinalphos, and Dichlorvos, along with 4 organocarbamate pesticides:
Aldicarb, Carbaryl, Carbofuran, and Carbosulfan.
Time: 30 minutes
Working Principle of the method 25
11
The method, a visual colorimetric biosensor, was developed through the careful
selection and optimization of the indicator, Bromothymol Blue. This indicator was
seamlessly integrated with the acetylcholinesterase assay for calibration based
on inhibition. A color code consisting of blue and green was meticulously
optimized and calibrated to correspond to specific pesticide concentrations. 5
The reaction occurred as follows:
Tube A: In Tube A, Acetylcholinesterase enzyme (AChE) was present. The
inhibition of AChE by pesticides can vary depending on the specific pesticide and
its concentration. However, AChE is not inhibited by water.
Tube B: In Tube B, the reaction involved the substrate acetylthiocholine iodide 10
(ATCI) and AChE in the presence of water. The reaction can be represented as
follows: ATCI + AChE + H2O ? Thiocholine + Acetate + AChE (hydrolysis
reaction) Here, ATCI acts as the substrate for AChE, and the enzyme catalyzes
the hydrolysis of ATCI, resulting in the production of thiocholine and acetate. The
presence of the indicator bromothymol blue allows for the detection of the 15
hydrolysis reaction.
Color Change
Water present: The color change was observed from blue to green in the presence
of the hydrolysis reaction. The bromothymol blue indicator underwent a color shift,
indicating the formation of thiocholine and acetate. 20
Pesticide Present: In the presence of a pesticide, the reaction with ATCI and
AChE does not undergo hydrolysis. Therefore, there is no production of
thiocholine and acetate. As a result, there is no change in the color of the solution,
and the blue color persists.
Preparation of Tube A and Tube B 25
12
Tube A
Tube A: Acetylcholinesterase enzyme (0.02U/µl)
Requirement
50 mM Sodium phosphate buffer (pH 7.0), and Acetylcholinesterase enzyme
For Stock Enzyme (1 U/µl) 5
Total enzyme concentration = 1000 U
Mix 1000 µl of 50 mM Sodium phosphate buffer (pH 7) in the enzyme vial to get
the stock enzyme solution with concentration of 1U/µl.
For Stock Enzyme (0.02 U/µl)
Take 10 µl of enzyme stock solution & add 490 µl of 50 mM Sodium phosphate 10
buffer (pH 7) to get 500 µl of enzyme working solution with concentration of 0.02
U/µl.
Add 30 µl working enzyme solution into the Tube A and stored in -21ºC.
Mathematical Calculations
50 mM Sodium phosphate buffer (pH 7.0) (For 100 ml) 15
To prepare 50 mM Sodium phosphate buffer (pH 7.0) for 100 ml:
Disodium phosphate (HNa2O2P.5H2O) quantity: 1.080 gm
Single sodium phosphate (NaH2PO4) quantity: 599.9 mg
Add the calculated amounts of disodium phosphate and single sodium phosphate.
Ensure that the pH of the solution is maintained at 7. 20
13
Adjust the volume to 100 ml using milli-Q water.
Tube B
Tube B was prepared with the following components and requirements:
50 mM Sodium phosphate buffer (pH: 7.8)
100 mM Acetylcholinethioiodide 5
Bromothymol Blue
Combine the following components:
450 µl of 50 mM Sodium phosphate buffer (pH 7.8)
50 µl of Acetylcholinethioiodide (100 mM)
50 µl of Bromothymol Blue 10
Mix the components thoroughly to ensure homogeneity.
Store Tube B at -21ºC for further use.
By following these steps, Tube B was prepared with the specified volumes of
sodium phosphate buffer (pH 7.8), acetylcholinethioiodide, and bromothymol
blue. The solution was then stored at -21ºC for future use. 15
Reaction for Pesticide Residue Detection
To perform the pesticide detection test using Tube A and Tube B, the following
steps can be followed (Figure 1 (a & b)
Prepare Tube A as described previously, containing 30 µl of the working enzyme
solution (0.02 U/µl) stored at -21ºC. 20
Add 100 µl of the pesticide sample to Tube A.
14
Incubate Tube A, with the pesticide sample, for 10 minutes at an appropriate
temperature for the enzymatic reaction to occur.
After 10 minutes, transfer the entire solution from Tube A into Tube B. Tube B
contains 450 µl of Sodium phosphate buffer (pH 7.8), 50 µl of
Acetylcholinethioiodide, and 50 µl of Bromothymol Blue. 5
Allow the combined solution in Tube B to incubate for 15 minutes. During this
time, observe any color change that may occur.
If the color changes from blue to green, it indicates a negative result, meaning the
pesticide concentration in the sample is below the specified threshold.
If the color remains blue and does not change, it indicates a positive result, 10
suggesting that the pesticide concentration in the sample is equal to or above the
specified threshold.
For confirmation, wait an additional 15 minutes and observe the color again. If the
color remains unchanged after this confirmation period, the positive result is
confirmed. 15
Experiments
Experiment 1: Pesticide Residue Detection
In this experiment, the objective was to detect the presence of the pesticide
carbosulfan in various samples, including negative and positive controls. The
detection minimum residue limit (MRL) for carbosulfan was set at 0.01 parts per 20
million (ppm). The experiment included several samples, a negative control, and
positive control samples with known concentrations of carbosulfan.
Negative Control Samples
Positive Control
(Carbosulfan)
15
1. Milli-Q Water 3. Green Mango 6. 0.01 ppm
2. Carbosulfan
(0.008 ppm)
4. Chinese Okra 7. 0.02 ppm
5. Chilli 8. 0.04 ppm
9. 0.08 ppm
Results
The results of the experiment indicated confusion in the interpretation of the color
changes observed. It is mentioned that there was confusion in the green-bluish
color, implying that the observed color changes did not clearly differentiate 5
between negative and positive results. It is also noted that a low enzyme
concentration was used, which may have contributed to the difficulty in
interpreting the color changes accurately. (See figure 2)
Conclusion
Based on the results and the difficulty in interpreting the color changes, it can be 10
concluded that further optimization of the experimental conditions, including
enzyme concentration, may be necessary to improve the accuracy and reliability
of the pesticide residue detection method. Additionally, considering the confusion
in color interpretation, further validation and confirmation tests may be required to
confirm the presence or absence of carbosulfan residues in the samples. 15
Experiment 2: Effect of Water & Time for Pesticide Leaching
Negative Sample (Tomato) Positive (Carbosulfan)
1. Distilled Water 2. Distilled Water (5 min) 8. 0.05 ppm
3. Distilled Water (10 min)
4. Distilled Water (15 min)
16
5. Tap Water (5 min)
6. Tap Water (10 min)
7. Tap Water (15 min)
Result
The results of the experiment indicated the following observations:
• No significant difference was observed between the different time intervals
(5 min, 10 min, and 15 min) in terms of pesticide leaching. 5
• The sample treated with distilled water showed a negative result,
suggesting the absence of carbosulfan residues.
• On the other hand, the sample treated with tap water yielded a positive
result, indicating the presence of carbosulfan residues. (See figure 3)
Conclusion 10
Based on the results obtained, it can be concluded that the time interval did not
have a significant impact on the leaching of carbosulfan from the tomato sample.
However, the type of water used for leaching showed a distinct effect. The distilled
water sample yielded a negative result, indicating the absence of carbosulfan
residues. In contrast, the tap water sample resulted in a positive result, indicating 15
the presence of carbosulfan residues.
Experiment 3: Effect of sample weight
In this experiment, the objective was to investigate the effect of varying sample
weights on pesticide detection. The negative control consisted of Milli-Q water,
while the samples were wheat grains to which charcoal was added. The positive 20
control contained a known concentration of the pesticide carbosulfan.
Negative Control
Samples (Wheat)
(Charcoal added)
Positive Control
(Carbosulfan)
Milli-Q Water 5 gm 7. 0.02 ppm
10 gm
17
15 gm
20 gm
25 gm
Result
The results of the experiment indicated the following observations:
• No pesticide was detected in any of the samples, including the negative
control and the wheat samples with varying weights. 5
• The absence of a color change or any other indication of pesticide
presence suggested that the samples were free from carbosulfan residues.
(See Figure 4 & 5)
Conclusion
Based on the results obtained, it can be concluded that no pesticide, specifically 10
carbosulfan, was present in the wheat samples. The addition of charcoal and
variation in sample weight did not show any significant effect on the pesticide
detection results.
Furthermore, the absence of a pesticide detection in the samples, including the
negative control, indicates that the experimental setup and methodology used 15
were effective in ensuring accurate detection of pesticide residues.
Experiment 4: Trial with different vegetables
In this trial, the aim was to detect the presence of pesticide residues in various
vegetables using a negative control, positive control (carbosulfan), and different
samples. 20
Negative Control Samples
Positive Control
(Carbosulfan)
1. Milli-Q Water 1. Plain Tori 1. 0.01 ppm
2. Carbosulfan (0.005
ppm)
2. Brinjal 2. 0.02 ppm
18
3. Carbosulfan (0.008
ppm)
3. Beans 3. 0.04 ppm
4. Capsicum 4. 0.08 ppm
5. Cucumber 5. 0.1 ppm
6. Bitter Gourd 6. 1 ppm
7. Tinda
8. Taro
9. Lady finger
10. Turnip
11. Spinach
12. Chilli
13. Green
coriander
14. Tomato
15. Tori
16. White Beans
Result
Based on the results obtained from the trial, it was observed that the bitter gourd
(6) sample showed a positive presence of pesticide residues, while no pesticide
residue was detected in the other vegetables tested. (see figure 6 & 7) 5
Conclusion
Based on the results obtained, it can be concluded that out of the tested
vegetables, only Bitter Gourd (6) was found to have pesticide residue. All other
vegetables showed no detectable levels of carbosulfan residues.
Experiment 5: Pesticide residue in tap water 10
In this experiment, the objective was to determine the presence of pesticide
residues in tap water. The negative control consisted of Milli-Q water, while the
sample used was tap water.
19
Negative Control Samples
1. Milli-Q Water 2. Tap Water
Result
The results of the experiment indicated the following observations:
• The negative control, Milli-Q water, showed no indication of pesticide
residue.
• However, the tap water sample tested positive for the presence of pesticide 5
residues.
(See figure 8)
Conclusion
Based on the results obtained, it can be concluded that the tap water sample (2)
contained pesticide residues. This finding suggests that the tap water used in the 10
experiment was contaminated with pesticides.
Experiment 6: Confirmatory test
In the confirmatory test, different control samples and positive controls were used
to validate the results obtained.
Negative Control Positive Control (carbosulfan)
1. Milli-Q Water 3. 1 ppm
2. Distilled Water
Result 15
A green color started appearing after 15 minutes and was confirmed within 30
minutes.
(See figure 9)
Conclusion
Based on the observed results, the green color change that occurred after 15 20
minutes can be considered a reliable indicator for detecting the presence of
pesticide residues. This indicates that the calculation and preparation methods
used in the experiment were effective in identifying and confirming the presence
20
of pesticide residues in the tested samples. The color change serves as a visual
cue to differentiate between samples with and without pesticide residues,
providing a straightforward and easily interpretable result.
Experiment 7: Sensitivity test and charcoal effect
• With charcoal 5
• Without charcoal
Negative Control Positive Control (Carbosulfan)
1. Distilled Water (Fresh) 5. 0.01 ppm
2. Distilled Water (Old) 6. 0.1 ppm
3. Milli-Q Water 7. 1 ppm
4. Carbosulfan (0.005 ppm) 8. 10 ppm
9. 20 ppm
Result
Charcoal interferes with pesticide residue analysis. (see figure 10)
Conclusion 10
The conclusion drawn from the experiment is that charcoal adversely affects
pesticide residue analysis. It has the ability to adsorb pesticides, thereby removing
them from the sample solution and causing inaccurate measurements. This
interference can potentially mask the presence of pesticide residues, leading to
false-negative results or reduced sensitivity in detecting low levels of pesticide 15
contamination.
Experiment 8: Different enzyme concentration
Negative Control Enzyme Concentration
1. Distilled Water 30 µl
2. Distilled Water 60 µl
3. Distilled Water 90 µl
Result
21
• After 15 minutes, the sample with 30 µl of enzyme experienced a significant
color change, transitioning from blue to green.
• After 10 minutes, the sample with 60 µl of enzyme underwent a noticeable
color change, shifting from blue to green.
• After 5 minutes had elapsed, the sample with 90 µl of enzyme experienced a 5
distinct color change, transitioning from blue to green.
(See figure 11)
Conclusion
From these observations, it can be concluded that the enzyme concentration has
an impact on the rate and intensity of the color change in the reaction. Higher 10
enzyme concentrations led to a faster and more pronounced transition from blue
to green, while lower enzyme concentrations resulted in a delayed or less
pronounced color change.
Experiment 9: Rechecking the reactions
The aim was to recheck the reactions and confirm the reliability of the color 15
change in both the negative control and positive control samples.
Negative Control Positive Control (Carbosulfan)
1. Distilled Water 2. 10 ppm
Result
22
• Negative Control (Distilled Water): After 15 minutes, a color change from
blue to green was observed.
• Positive Control (10 ppm Carbosulfan): No color change was observed
even after 15 minutes.
(See figure 12) 5
Conclusion
Based on these results, it can be concluded that the color change from blue to
green is specific to the presence of pesticide residues. The negative control,
representing the absence of pesticide, exhibited the expected color change,
indicating the reliability of the method. On the other hand, the positive control, 10
containing a known concentration of Carbosulfan, did not show any color change,
further validating the specificity of the colorimetric assay.
5.6. Summary of Experiments
BioKit pesticide residue detection is pH sensitive.
S. No. Experiments Result
1. Pesticide Residue Detection 1. Confusion in detection of blue?greenish colour.
2. Low enzyme taken.
2. Effect of Water & Time for
Pesticide Leaching
1. Only distilled water used in this
experiment.
2. No difference in time interval.
3. Effect of sample weight No change in weight wise.
4. Trial with different vegetables Bitter gourd (Karela) was positive
5. Pesticide residue in tap water Tap water was positive
6. Confirmatory test After 15 minutes colour change
(blue to green)
7. Sensitivity test and charcoal
effect
Charcoal not required
23
8. Different enzyme concentration Colour change (Blue to green)
1. 30 µl enzyme (15 minutes)
2. 60 µl enzyme (10 minutes)
3. 90 µl enzyme (5 minutes).
9. Rechecking the reaction After 15 minutes colour change
(blue to green)
Certain features of the invention have been described with reference to the
example embodiments. However, the description is not intended to be construed
in a limiting sense. Various modifications of the example embodiments as well as
other embodiments of the invention, which are apparent to the persons skilled in 5
the art to which the invention pertains, are deemed to lie within the spirit and scope
of the invention.
10
15
20 ,CLAIMS:WE CLAIM
1. A method to detect concentration of residual pesticides available in
fruits and vegetables comprising
I. Preparing a pesticide sample of fruit or vegetable and taking 80
- 120 µl of the same into a tube (A), 5
II. Adding 20 – 40 µl of enzyme solution into the tube (A) and
incubating the same for 5 – 15 minutes at room temperature to
obtain enzymatic solution,
III. Preparing a solution by mixing homogeneously 400 - 500 µl of
Sodium phosphate buffer, 40 - 60 µl of Acetylcholinethioiodide, 10
and 40 – 60 µl of Bromothymol Blue in another tube (B),
IV. Adding the enzymatic solution of the tube (A) into solution of
another tube (B) and incubating the same for 10 – 20 minutes,
observing color change of the solution for initial detection of
concentration of residual pesticides available in fruits and 15
vegetables,
V. Incubating the above mixture for another 10 – 20 minutes and
observing the color change of the solution for confirmation of
detection of concentration of residual pesticides available in
fruits and vegetables. 20
2. The method to detect concentration of residual pesticides available in
fruits and vegetables as claimed in claim 1, wherein the enzyme
solution is of 0.01 – 0.03 U/µl of concentration and pH value between
6.8 – 7.2.
3. The method to detect concentration of residual pesticides available in 25
fruits and vegetables as claimed in claim 1, wherein the sodium
phosphate buffer is of a concentration 45 - 55 mM sodium phosphate
buffer and pH value between 7.6 – 8.0.
25
4. The method to detect concentration of residual pesticides available in
fruits and vegetables as claimed in claim 1, wherein the pesticide
sample of fruits and vegetable is prepared by taking 80 - 120 grams of
the desired fruits or vegetables and cutting the same into small pieces,
placing the cut pieces into a container and adding 80 - 120 ml of water 5
into the container, shaking the container intermittently for 8 - 12 minutes
to facilitate the transfer of pesticides from the vegetable surface into the
water, allowing the mixture to settle for a few minutes, decanting
aqueous layer from the top of the container such that to leave behind
the solid fruits or vegetable material to obtain pesticide sample. 10
5. The method to detect concentration of residual pesticides available in
fruits and vegetables as claimed in claim 1, wherein the change of color
of the solution from blue to green, is indicated that the pesticides
concentration in the sample is below the specified threshold.
6. The method to detect concentration of residual pesticides available in 15
fruits and vegetables as claimed in claim 1, wherein the unchanged of
color of the solution, is indicated that the pesticides concentration in the
sample is above the specified threshold.
Dated this 1
st day of July, 2024.
20
R. P. Yadav
IN/PA – 954
of sr4ipr Partners
Applicant’s Attorney