1. Thesis submitted for the partial fulfillment of the degree
of
Bachelor of Technology in Biotechnology
by
Nikita Khaitan & Ritika
Under the guidance of
Prof (Dr.) SUBHABRATA SENGUPTA
Dean (PG & Research)
PROFESSOR, DEPARTMENT OF BIOTECHNOLOGY
DEPARTMENT OF BIOTECHNOLOGY
HERITAGE INSTITUTE OF TECHNOLOGY, KOLKATA

2. • Primarily attempt was made in the project to develop a
mercury detection kit which could determine the presence of
mercury in water sample at concentration below permissible
level of 1-2 ppb recommended by WHO.

3. • Mercury poisoning is a type of metal poisoning and a medical condition
caused by exposure to mercury or its compounds.
• Mercury has a number of effects on humans:
 Disruption of the nervous system
 Damage to brain functions
 DNA damage and chromosomal damage
 Allergic reactions, resulting in skin rashes, tiredness and headache
 Negative reproductive effects, such as sperm damage, birth defects and
miscarriages

4. • In the United States, both Food and Drug Administration (FDA) and the
Environmental Protection Agency (EPA) allow levels of total Hg2+ less than
0.002 milligram (mg)/L in both tap and bottled water.
• The Bureau of Indian Standards (BIS) has laid down safety limits for drinking
water at 0.001mg of mercury per litre (1 ppb).
• In India, some of the major rivers tested for heavy metals by the Industrial
Toxicological Research Centre (ITRC), Lucknow, were found to contain
mercury in alarming levels. Several studies on fish and prawns in Mumbai,
Kolkata, Orissa, etc, have reported alarming rates of mercury concentration.

5. • Physical methods use techniques like: XRF, AAS, AFS, etc.
• Also there are many enzymatic methods that use: glucose
oxidase, papain, bromelain, and many other proteases, for the
detection of mercury ion.
• The main principle behind using these enzymes is that mercury is
well known for its ability to react with sulfhydryl (thiol ) groups
of proteins, which are frequently responsible for enzyme’s active
center.

6.  All these instruments are very costly.
 These require high technical skills to be operated.
 These are not ultra-sensitive.

7. A flat bottom flask
Immobilized starch
strips of dimension:
LENGTH=2.8cm
HEIGHT=1.4cm
Test tube + starch strip
Materials used for KIT development:

8. • A non-specific glucan hydrolase (amylase) enzyme was used,
extracted from a plant Tinospora cordifolia.
• The Tinospora amylase was found to be highly sensitive to Mercury
ion, the most dangerous heavy metal ion present in drinking water.
• The property of mercury of inhibiting enzymes by binding at their
active site has been exploited to develop this kit.
• Starch-iodine test is employed for visible detection of mercury ion
in the water samples.

9. During hydrolysis of starch, one reducing group (aldehyde)
group is released by the cleavage of one glycosidic linkage.
The aldehyde group generated reduces dinitro salicylic acid
into diamino salicylic acid (red colour).The red colour is
quantitatively estimated at O.D 540 nm compared to glucose.
Thus OD reading is equated to moles of glucose equivalent
released during hydrolysis.

10. • 1% starch solution was prepared and enzyme was prepared for
usage.
• 1 ml starch was added to all test tubes.
• 10, 20, 30 µl of enzyme was added to 3 test tubes and volume
of each was made 2 ml by adding required amount of buffer.
• 3 ml DNSA was added to the control and all test tubes were
incubated at 50ºC for 30 minutes.
• DNSA was added to rest of the test tubes and heated for 10
minutes.
• O.D. was taken at 540nm.

11. 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 500 1000 1500
O.D.valuesat540nm
Amount of glucose(µg)
Amount of glucose vs O.D.
Values at 540nm
Linear (Amount of glucose vs
O.D. Values at 540nm)

12. • 1% starch solution is prepared.
• Different amount of starch is added to a number of test
tubes and rest of the volume is made upto 1 ml by
adding water.
• Iodine is added to each and certain amount of blue
colour is obtained in each.
• O.D. is taken at 640nm.

13. Iodine is known to remain entrapped in helical structure of long
chain starch and gives a very intense blue colour at very low
concentration. Hydrolysis of starch by amylase causes loss of
helical structure with the release of iodine and loss of blue colour.
0
0.2
0.4
0.6
0.8
1
1.2
0 0.2 0.4 0.6 0.8 1 1.2
O.D.valuesat640nm
Amount of starch(ml)
Amount of starch vs O.D.at
640nm
Linear (Amount of starch vs
O.D.at 640nm)

14. • 0.6% starch solution was prepared.
• Different concentration of this solution were made.
• Many filter paper strips were prepared and they were
soaked in different starch concentrations.
• They were then dried and added to the flat bottomed test
tubes.
• Buffer was added to the test tubes so that it covered the
strip.
• These were then incubated for 30 minutes and then
iodine was added.

15. IMMOBILIZED STARCH STRIPS OF DIFFERENT
CONCENTRATIONS GIVE DIFFERENT INTENSITY OF COLOUR
WITH IODINE.

16. • 0.6% starch solution was prepared
• Test tubes with immobilized starch strips are taken and
water was added.
• Different amount of enzyme was added to each test tube
and incubated.
• Iodine was then added to each.

17. DIFFERENT AMOUNTS OF ENZYME GIVE
VARYING AMOUNT OF COLOUR WITH IODINE.
100 100

18. TEST TUBE NUMBER MERCURYCONCENTRATION
I 10 mg/ml
II 1 mg/ml
III 0.1 mg/ml
IV 0.01 mg/ml
V 1 μg/ml
VI 0.1 μg/ml
VII 0.01 μg/ml
VIII 0.001 μg/ml = 1 ppb
IX 0.0001 μg/ml = 0.1 ppb

19. 5 6
7 8 9 B1
Thus, mercury was detected to the level of 0.1-1 ppb (i,e. till 8th dilution).

20. Samples Collected

21. • Industrial effluent (HALDIA)
• CME (college)
• CBT (college)
• Howrah station
• Sreerampore municipality
• Canteen tap (college)
• Hostel drinking water
• Canteen drinking water cooler
• Labour canteen (college)
• Macha (outside college)

23. • The enzyme- inhibitory method described for the detection of mercury ion
has been found to be very sensitive (less than 1 ppb) , much more potent
than all other methods as it is:
 very simple
 visually detectable
 no costly enzyme or reagents required
 can be carried out outdoors as well as in a very simple laboratory.
• This mercury detection kit will be very useful in confirming the presence
of mercury on the first stage. Thus, it will ensure safety from drinking
contaminated water. On the next level, quantitative estimation can be done
using specific equipments.

24. • Mukherjee A, Sengupta S, Ray L and Sengupta S, Evaluation of Tinospora
cordifolia Amylase as a Commercial Digestive Enzyme of Plant Origin,
J.Herbs Spices and Medicinal Plants, 18, 2012, 58-76.
• Mukherjee A, Ghosh AK and Sengupta S, Purification and
Characterization of a Thiol Amylase over Produced by a Non-Cereal Non-
Leguminous Plant, Tinospora cordifolia, Carbohydrate Res, 345, 2010,
2731-2735.
• Singh SS, Pandey SC, Srivastava S, Gupta VS, Patro B, Ghosh AC,
Chemistry and Medicinal Properties of Tinospora cordifolia, Indian
J.Pharmacol, 35, 2003, 83-91