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A PROJECT REPORT

ON

STUDY OF BIOSENSORS
(WITH AN EMPHASIS ON THE REACTIONS OF
DDT TO DETECT ITS PRESENCE)

GROUP MEMBERS
1) DIVYANK SHEKHAR (2015A3PS335H)
2) SANYAM JAIN (2015A7PS094H)
3) HANISH SARGIYA (2015A8PS479P)

PREPARED FOR GUJARAT ENVIRONMENT


MANAGEMENT INSTITUTE (GEMI)
ACKNOWLEDGEMENT

The Practice School programme of BITS Pilani has given us


the space to undertake some exciting projects along with the
exposure to the functioning of an autonomous organisation.
Our journey through this enlightening training programme
would not have been possible without the help of our learned
mentors. We would like to express our gratitude towards them
here.
First, we want to thank our instructor Dr. Ashish Chittora,
who was always ready to help us with his skillset and
regularly monitored our progress.
The vision and efforts of Dr.Sanjiv Tyagi, director, GEMI
were extremely conducive towards the betterment of the
project. His utilitarian approach and invaluable feedbacks
continuously guided us in the right direction.
Rupal Maam, our project instructor gave us useful feedbacks
and provided numerous papers, presentations and research
works to aid us in our quest to understand biosensors better.
Finally we would like to thank Nitasha Khatri Maam, our
fellow interns at GEMI and numerous other instructors and
officials who created such a supportive atmosphere for
constructive work.
INDEX
SNO TOPIC PAGE
1. ACKNOWLEDGEMENT 2
2. ABOUT GEMI 4
3. BIOSENSORS A BRIEF 5
OVERVIEW
4. PROJECT TITLE, 6
DESCRIPTION
5. TYPES OF BIOSENSORS 7
6. BASIC MODEL 8
7. TYPES OF MICROBIAL 9
REACTIONS
8. DDT AND ITS REACTIONS 10
9. THE pH PROBE CIRCUIT 12
10. REMAINING WORK 14
11. APPLICATIONS 14
12. OBSERVATIONS 15
13. INDIVIDUAL 16
CONTRIBUTIONS
14. REFERENCES 17
ABOUT GEMI

Gujarat Environment Management Institute is an


autonomous institute set up under the Forest department
of the government of Gujarat. It was established on the
lines of NEERI, when a need for regulating and studying
the impact of development on environment was given
emphasis.
It was registered on 1st March 1999 under the Bombay
Public Trust Act, 1950.
The institute has fully-fledged labs for testing the quality
of air, water and soil. These labs are recognised under the
Environment Protection Act, 1986.

MISSION STATEMENT - To Promote Conservation,


Protection, and Management of the Total
Environment through Scientific and Technical Pursuits in
order to maintain or restore the pristine elements of such
Environment
GEMI guides and regulates the monitoring of
environment in Gujarat. It carries out environmental
impact assessment activities before the establishment of
an industry in any particular area.
It also carries out research and development activities for
pollution control and analyses air, water and soil quality
in the nearby areas. Numerous journals and research
works are also published on a regular basis by the
institute.

BIOSENSORS A BRIEF OVERVIEW

In the most general terms, a biosensor is a device which uses


microbes or biological molecules like enzymes or antibodies
to detect the presence of chemicals. The microbes and living
organisms display certain reactions that may generate
physically measurable quantities. Certain types of enzymes
react with only specific types of pollutants, and these can be
used as confirmatory tests for the detection of these pollutants.
The physical parameters that change are sensed. These
include changes in pH, temperature, mass, electrical
conductivity etc.
The biologically sensitive elements can also be created
by biological engineering. The recognition component, often
called a bio receptor, uses biomolecules from organisms or
receptors modelled after biological systems to interact with
the analyte of interest. This interaction is measured by the bio
transducer which outputs a measurable signal proportional to
the presence of the target analyte in the sample. The general
aim of the design of a biosensor is to enable quick, convenient
testing at the point of concern or care where the sample was
procured.

PROJECT ALLOTED AND ITS


DESCRIPTION

We have been assigned work on biosensors, its applications,


and development of a basic design keeping in view its
usefulness to detect the presence of pollutants such as
DDT.
Different types of biosensors have been developed for some
specific enzymes. Still the challenge remains to develop
accurate and reliable ones for the detection of several other
pesticides including DDT.
Through our project, we take a look at the different types of
biosensors available currently. We then study the reactions of
DDT and the feasibility of making a biosensor to detect its
presence in a sample. Though most studies generally give a
brief description of the measurable and useful reactions for
development of biosensors, we attempt to design a sensing
and amplification circuit at a basic level in our project.
We will also attempt to link the electrical circuit for sensing
the pollutants through a microprocessor board such as arduino
for processing and display so that the results can be easily
analysed.
It is also necessary to calibrate the results obtained from self-
developed biosensors with that obtained from generally
available methods of detection, to realize the accurateness of
the measurements.

TYPES OF BIOSENSORS

Different types of Biosensors have been developed for


detecting the presence of specific categories of pollutants. The
development of a biosensor to detect DDT would have to
based on one of these designs, so it is important we take a
look at these.
Electrochemical measures the electronic current by
changes in ionic concentration between bio electrodes.
Amperometric Biosensor - Based on the movement of
electrons. A normal contact voltage passes through the
electrodes to analyse. An alternate current flow can be also be
measured. Current depends on concentration of substrate. The
Clarke oxygen electrode is a common example of this. This
has been used in the detection of a variety of organophosphate
containing pesticides, with different specific functional groups
reducing oxygen to H2O2 in the presence of enzymes.
Potentiometric Biosensor - This type of sensor measures
potential difference instead of current. This potential
difference is generally directly proportional to the
concentration of the substrate. The major limitation of
potentiometric biosensors is the sensitivity of enzymes to
ionic concentrations such as H+ and NH+4.
Several other biosensors have been developed which include
optical and piezoelectric biosensors, but we will focus mainly
on the above three sensor types as they require lesser
sophisticated techniques for their development than the latter
ones.

BASIC MODEL DEVELOPED

In a very general view, the biosensor would consist of three


basic components:
BIOSENSORS Bio receptors + Bio transducers +
processing device.
The bio receptor can be anything that reacts with the analyte
to be tested, and generates a measurable signal in any form
that can be recognised by the corresponding type of
transducers, as discussed in the above section on Types of
Transducers.
A bio transducer will convert this signal into analog or digital
output in the form of voltage or current. The changes
occurring during the reactions utilised may cause changes that
alter the conductivity of solutions, resulting in voltage
changes. Since we are working with reactions of the order of
micro levels and concentrations of the order of parts per
billion, we will have to amplify the signals at the beginning,
preferably using operational amplifiers or MOSFETs.
The arduino board will then be uploaded with a code
containing the data of different reactions, and will
correspondingly display which pollutant is present in the
sample based on the voltage fluctuations of the transducer.

TYPES OF MICROBIAL REACTIONS


While the type of biomolecule used can vary widely,
biosensors can be classified according to common types of bio
receptor interactions involving:
a) Antibody Antigen Interactions- key and lock
system where specific antibody binds to specific
antigen and changes can be seen by tracers like
fluorescent molecules or radioisotopes. That specific
antibody will not react in the same way with any other
antigen, so this reaction calls for high specificity and
increased conformity.
b) Enzymatic interactions Specific enzymes may
convert the analyte into products that can be measured
or they may inhibit a general reaction, with a change
in expected values indicating that a reaction has taken
place with that enzyme. In our project, we found an
enzyme called DDT hydrolase which acts upon DDT
and is found in houseflies.
c) Several new techniques utilizing reactions of different
biological elements are being developed. These
include artificially binding proteins or DNA
molecules. These require sophisticated techniques and
we shall concentrate mainly on the first two
interactions in relation with observing the possibility
and feasibility of measuring DDT levels with
biosensors.

DDT AND ITS REACTIONS

1) We were asked by the institute to look into the


possible reaction of DDT with plasmodium to utilize
the reaction for the bio receptor, as DDT kills the
mosquitoes carrying plasmodium. DDT is moderately
toxic, and has potent insecticidal properties; it kills by
opening sodium ion channels in insect neurons,
causing the neuron to fire spontaneously. This leads to
spasms and eventual death. So we found that it does
not interact with plasmodium directly.
2) Degradation from soil Microorganisms-
Soil contains microbes which can degrade DDT to
DDD. Six bacteria isolates belonging to genera
Bacillus, Staphylococcus and Stenotrophomonas
were identified, and a mix of these isolates
increased the rate of degradation.
DDD
No microbes have been found that degrade DDT as
a carbon/energy source, but some might degrade
organochlorine.

THE PROBLEM HERE is that the isolation of


soil containing these microbes and their incubation
process would take more than a month, with
different samples collected from different areas
around the world. The testing and degradation of
DDT by these microbes will further take a month.
But this degradation opens a possible reaction
mechanism which may be further investigated.
3) Dehydrochlorinase enzyme found in houseflies
converts DDT to DDE.

DDE(DichloroDiphenylEthylene)
This reaction mechanism can also be explored for
isolating enzymes to detect DDT in a water sample.
Other reactions of DDT that produce HCl were observed but it
is difficult to measure its presence in water with these as the
reactions take place in extreme conditions owing to the
stability of DDT. But having estimated a measure of the order
of reactions, we can now proceed towards developing a
transducer circuit that is capable of detecting signals of such
low levels.

THE pH PROBE CIRCUIT

This is a very basic


diagram of a pH meter. The pH probe shown consists of a
silver electrode acting as a reference electrode and a glass
electrode. We have to develop it such that the oxidation
reduction reactions that take place here sensitive enough to
respond to changes in the pH during reactions of pollutants at
the microbial level. Thus a very simple solution can be to
insert cascaded amplifiers between the pH probe and the
TL082 operational amplifier. This particular device gives us
a gain of 17V/V and the entire circuit is calibrated to give the
vout on a logarithmic pH scale from 0 to 14. This Vout is
directly proportional to the pH and the values can be read
from a voltmeter.

A major concern here is whether the pH probe would be able


to detect such small changes during the course of important
Reactions of the analytes. We researched for some possible
methods for detection and came across the following methods:
1) Using nanotechnology to get detectable current signals.
According to the Gap + Width rule, 95% of the current
passes below the distance that equals the Pitch (W+G) of
the connected electrodes. Since most of the reactions of
our interest occur in the range of 10 to 100nm, having a
pitch of similar dimensions can maximize sensitivity.

2) Other detection techniques are available which include


use of gold plated Nano particle films and a concept of
realizing a capacitor between the electrode and ions in
the solution itself.

These methods require advanced and expensive technologies,


some of which are still in development mode. We will have to
compare the cost of manufacturing these and the increase in
the accuracy of measurements these will offer.

REMAINING WORK

We have to work towards finding a suitable reaction for DDT


so that its presence can be detected. The same model will also
aid in the detection of numerous other pesticides as well.
An amplifier circuit between the pH probe and the
operational amplifier has to be installed. This will be based on
the order of reactions of DDT, and so its specifications are
dependent on the biological tests performed by GEMI.
At last the arduino has to be supplied with a simple code for
displaying which pollutant is present based on the voltage
fluctuations in the pH probe circuit.

APPLICATIONS OF BIOSENSORS

Biosensors are a rapidly emerging technology finding


applications in various sectors. Recently, glucose monitoring
biosensors based on amperometric detection of glucose levels
were introduced and proved to be highly successful. Similar
applications can include checking the food quality. It also
finds applications in other health related areas such as
detection of cancer affected cells.
These can be employed to check ozone levels in the
atmosphere as well. The advantage that this technology offers
is of increased precision of results.
The most extensive use of biosensors has been in the detection
of pesticides, mostly in water samples from different water
bodies. The amperometric technique allows the detection of a
large number of organophosphorous insecticides and
pesticides.
A biosensor that is capable of detecting DDT would be of
tremendous importance to the research and health sector.
Although the regular trading of DDT is only permitted under
constraints, large quantities of it deposit in soil, water bodies
or in the bodies of animals as DDT spraying is still common.
Thus biosensors have an increasing scope in the field of
research currently.

OBSERVATIONS

There were several interesting points that we noted during the


course of our study on biosensors and the reactions of DDT.
Some of these worth mentioning are:
1) It is very feasible to develop noise level sensing
transducers on similar grounds as biosensors. Several
types of such low cost transducers installed at different
places will help administer noise pollution more
efficiently.
2) The attachment of bio receptors (like enzymes,
antibodies) on the surface of transducers using advanced
surface engineering concepts (layer by layer deposition
using xerogel) can increase the sensitivity of the system
to a great extent.
3) The feasibility of developing a biosensor for detecting
DDT should be compared with the cost required for its
detection with current technologies and the difference in
precision levels in detection.
4) The circuit can be tested on virtual circuit boards and
simulated there itself to prevent the cost of additional
equipment, which might not be of use later in the
project.

INDIVIDUAL CONTRIBUTIONS

Divyank Shekhar
Reactions of DDT and other reaction mechanisms that
can be used in developing biosensors.
pH probe circuit and study of small scale detection of
pollutants.
Preparation of amplification circuits and current methods
of DDT detection
Sanyam Jain
Surface engineering requirements for increasing
sensitivity of the transducers.
Advantages, disadvantages and working of
potentiometric transducers.
Research on other types available sensor technologies
and acoustic transducers.
Hanish Sargiya
Comparison between different types of sensors with a
look at their negatives.
Study on amperometric and optical methods of detection
of substrates.
Preparation of a model for biosensors with arduino
board.

REFERENCES

http://www1.lsbu.ac.uk/water/enztech/biosensors.html

https://www.edgefx.in/biosensors-types-its-working-and-applications/

https://en.wikipedia.org/wiki/Biotransducer

http://www.learningaboutelectronics.com/Articles/Sound-detector-
circuit.php
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Quesada-Gonzlez, Daniel; Merkoi, Arben (2016). "Mobile phone-based
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https://damien.douxchamps.net/elec/ph_meter/
http://www.instructables.com/id/cheap-DIY-electronic-pH-meter/