DEVELOPMENT OF KIT FOR ULTRA-SENSITIVE DETECTION OF MERCURY IN DRINKING WATER SAMPLE

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 BIOTECHNOLOGYHERITAGE INSTITUTE OF TECHNOLOGY, KOLKATA

Objective

 • 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.

Introduction• 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

Facts

• 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.

Mercury Detection

• 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.

Demerits of existing methods

All these instruments are very costly.

These require high technical skills to be operated.

These are not ultra-sensitive.

Ultra-sensitive Mercury detection

A flat bottom flask

Immobilized starch strips of dimension: LENGTH=2.8cm HEIGHT=1.4cm

Test tube + starch strip

Materials used for KIT development:

Working Principle

• 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.

Principle of Amylase Assay

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.

Amylase Assay Protocol• 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.

Standard curve of glucose

100 200 300 400 500 600 700 800 900 100011000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Amount of glucose vs O.D. Values at 540nm

Linear (Amount of glucose vs O.D. Values at 540nm)

Amount of glucose(µg)

O.D

. val

ues

at

540n

m

Standardization of starch-iodine colour

• 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.

Standard curve of iodine

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.

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.10

0.2

0.4

0.6

0.8

1

1.2

Amount of starch vs O.D.at 640nmLinear (Amount of starch vs O.D.at 640nm)

Amount of starch(ml)

O.D

. val

ues

at

640n

m

Determination of minimum starch concentration

• 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.

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

Determination of minimum enzyme concentration

• 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.

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

100 100

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

Mercury concentration at different dilutions

Determination of minimum mercury concentration

5 6 7 8 9

B1

Thus, mercury was detected to the level of 0.1-1 ppb (i,e. till 8th dilution).

Samples Collected

Different Water Samples

• 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)

Results of the Test

Discussion & Conclusion• 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.

References

• 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

THANK YOU !!