Dear students, welcome to the second lecture
on Biosensors. Today in this particular lecture,
we are going to discover; we are going to
recover different kinds of ah different kinds
of Biosensors and what differentiates them
from each other and how we have utilized a
wide array of physical and chemical properties
to be able to understand; what kind of contaminants
are present in an environment and to get rapid
information. So, that we can take quick actions;
we can make quick well directed in informed
interventions to tackle environmental health
and public health problems.
So, let us get started with Biosensors already
as a recap.
Remember, what Biosensor is; here you have
your analytes in the water or in here, we
have analyte in water or in sample . So, these
are your targets and then here you have bio
receptors. So, if this is the shape of an
enzyme by the way. So, you have bio receptors
and you it so happens that your target is
a perfect match for your receptor.
For your bio receptor when it matches, it
undergoes conformational change change in
physical properties chemical properties or
so and so forth and when it undergoes change;
this change can be converted into an electronic
signal, this entire business is part of job
of transducer and then the electron electronic
signal can be container replay can be in output
as either information on a screen or as a
light like red light not good green light
good something like that or it can be converted
read by a computer which gives us meaningful
data already.
Now, where is and where our sensor is important.
What is the scope for them in the market and
how would this help you in your career in
your entrepreneurship well sensors have at
least 3 very well recognized markets. One
is the diagnostic market, this is a very well
established, very large market in our country
and we have a lot of foreign players entering
our market right now, selling our public health
bodies, one of the a most advanced Biosensors
and sensors we have for detecting; public
health environmental health challenges.
So, there is lot of the scope for generating
developing new sensors for the diagnostic
market. Now when you talk of diagnostic market
basically we are trying to get an idea whether
ah the contaminant is present or not where
the target is not present or not.
So, this is diagnosis and diagnosis is not
limited to hospitals and which is the second
point actually clinical testing which is diagnosed
diagnosis in context of hospitals or clinics.
So, definitely diagnostic market for example,
I want to know whether a particular milk has
a particular protein or not; what does it
have a prote a one protein or does it not
have I might be interested to know that after
I have done a particular experiment on potato
its a particular proteins present or not.
So, all these kind of diagnoses can be done
using your Biosensors and there is a big market
for it and when this kind of diagnosis is
done in hospital clinical setting you have
clinical testing and this is no doubt there
is biggest diagnostic markets, then there
are other markets and nearly ah ah even even
the multi medical arena, we have a lot of
ah diagnostic market and agriculture in veterinary
soap and paramedical sciences and no wonder
definitely environmental sciences already.
So, what are the requirements of sensor of
what kind of sensors do we require? There
should be relevance of output signal to measurement
environment.
So, whatever I am measuring in the environment
whatever kind of environment I am in the output
that I give should be relevant to it next
is accuracy and repeatability it should not
be. So, that once this sensor gives me a value
of two next time it gives me 200 and then
gives me 2 crore, I have no idea what to believe.
So, it should be accurate and it should be
repeatable and the accuracy is the difference
between accuracy and repeatability perhaps,
but what we are mentioning here is accuracy
and precision. So, not only should I get same
similar values every time I take measurement,
but also it should be close to the actual
value of the true value which is accuracy.
Next is sensitivity and resolution. So, sensitivity
is the ability to detect lower amounts of
analyte; for example, if I have only 10 to
power 2 copies gene copies of vanA vancomycin
resistant gene per ml which is very very low
concentration. If I am able to detect it,
then I have high sensitivity. So, sensitivity
is; what is the lowest concentration you can
detect and get a good signal. Now resolution
especially if you are how clear is your signal
as if we have multi analyte system, if there
is a Biosensor that can detect glucose it
conducted sucrose fructose and other kinds
of sugar pentose then how well it can tell
me well the detection I am getting is of pentose
not of fructose for example, if I have a sensor
that measures that detects mrsA it detects
vanA like we talked about in the last class
and it also detects bla-1 gene better lactamase
a 1 gene.
Now, so, very good I have a sensor that can
detect all 3 is really bad antimicrobial resistant
genes in one go, but let us say it gets a
signal, yes, I have a signal how well can
it tell me with what confidence and clarity
can it tell me that this signal belongs to
bla-1 and not vans and mrsA.
So, this theory t; it comes with the resolution.
So, we definitely want to buy a sensor to
have very high sensitivity which means very
low detection and quantification levels and
very high resolution. Next is dynamic range,
then we have speed of response we have talked
about this in previous lecture two that we
want our Biosensors to give us as rapid data
as possible specially when we are suffering
from public health epidemics and we have serious
environmental challenges to take care of.
Next is in sensitivity to temperature or temperature
compensation now one thing is there in country
like India and many countries around around
the world we have temperature variability
in both the diurnal cycle and also seasonal
cycle. So, for example, where I am right now
in north India in winter the temperature might
fall down in you I am right now the temperature
might fall down to below 5 degree Celsius
and then at in during summer; we we might
stay consistently 46 degree Celsius plus which
is quite a big range of temperature. So, my
sensor should not fail at these extreme ends
it should not go at 48 degree; 49 degrees
Celsius or should not stop working at very
low temperatures.
Now this brings me to a very important point
we are talking about Biosensors right. So,
Biosensors have biological agents that act
as a receptor now for example, have enzyme,
but this enzyme is only very specific at particular
temperature you increase the temperature denatures
and you can detect anymore. So, this is one
limitation with certain kinds of Biosensors.
So, Biosensors this is very very important,
it should not be sensitive to temperature
even as temperature is changing, it should
be very reliable and even if the data moves
with temperature, let us set overestimates
at higher temperature or underestimates at
higher temperature there should be a way for
us to do temperature compensation there should
be a way for us to correct it mathematically
reliably which brings me to another point.
I hope you can see this here if you are talking
about temperature changes we are not expecting
our Biosensors to be sitting in control temperature
rooms laboratories we are ideally looking
for sensors that we can actually carry take
them to environment get the data right there
and right then that is the demand of today.
So, for that we really need that our ah our
sensors should be insensitive to temperature
or should have ability for temperature compensation
next is they should be insensitive to electrical
and other environmental interference.
For example let us say in lab you had a sensor
to detect mrsA, vanA and bla-1 gene very good
it worked perfectly. Now, you go to upper
finger canal and over here you go to river
Yamuna or you go to river Tapi. Tapi is very
clean, you go to other rivers that are in
really bad state and then what you notice
is that it is given its going haywire your
sensor is giving you data that does not make
sense why perhaps because it is quite possible
actually, it is quite possible that you have
other interfering agents that are not able
to give you good data, it is also possible
for examples if you have a magnetic based
ah magnetic base for ph base or conductivity
bases sa conductivity based transducers then
we have electrical interference and then things
might go haywire and we might not get reliable
data. So, regardless of what is happening
in the environment regardless of the temperature
your Biosensors should be able to give very
reliable information.
Now, let us look at the earth one amenable
to testing and calibration should be very
easy for us to test whether it is giving the
right date or not it should be easy for us
to test the value of inner sample, we should
also have the ease of calibrating it; for
example, most of the students here; in this
class would have at some point in their life
than ph analyses which is basically dipping
the ph electrode and finding out the ph of
the liquid now if you remember ideally before
you dipped in your ph electrode into the sample
you calibrated it using ph four and depending
on what kind of analysis you are doing a higher
ph solution which you know is a ph four or
at a higher value of ph. So, once you have
2 calibration point; you can be more sure
about the data that you are going to get.
So, it should be amenable to calibration.
Next with reliability and self checking capability
again same thing over and over again I should
be able to trust the data because it is repeatable
its accurate and it has shown its middle over
and over again we should have a self checking
capability it should be able to run auto check
and find out what the problem is if it is
working fine or not. So, if there is a problem,
but I do not detect; let us say I am in the
field and I mention ph is equal to nine of
water and I am surprised, but I am like ah
the instrument is very reliable and I would
record it and then we informed the ah civilians
and the government agencies and people get
concerned; however, if the sensor has a self
checking capability it can say wait something
is wrong with me take me to the mechanic first.
So, that kind of information is helpful.
Next is physical robustness again as I was
mentioning earlier that our target perhaps
now is not to have instrument sophisticated
giant sensors that sit in the labs national
laboratories regional laboratories, but we
want sensors that we can actually carry in
the field carry in the villages and get the
data for that; it is very important that the
sensor should have physical robustness lets
I need to ship the sample the sensor from
Chennai to Delhi in order to do that it will
go by air mail or by a train mail; however,
it go by road; however, it is transported
from Chennai to Delhi if it does not have
physical robustness; it is it is going to
arrive damaged and that is going to be a loss
to economy and also loss to environment in
public health because we cannot rotate contaminants
that we could have detected by using it and
thus physical robustness is very important.
Also for example, I am working with Namami
Gungi and I take my sensor and I take it to
my village which is some 30 kilometers from
here and by the time; I reach there because
the village roads are not very good my sensor
is damaged that we do not want next our service
requirements that service requirements should
be minimal and the service should be very
simple because we noticed that in many parts
of our nation many labs many regions many
research facilities servicing annual maintenance
and ah is very challenging.
Next capital cost; obviously, we do not want
it to be very expensive and running cost in
life he wanted to have higher life and the
older and running clock cost and the acceptability
by user it should be something that people
want to use and then product safety sample
whole system must not be contaminated by sensor.
So, ah not this is very important. So, the
safety of the sample safety of the host is
very important sensor should not put should
not endanger the health the well being of
the person; who is doing the sa also the innards
of the sensor should not be damaged on exposure
to a particular sample already.
So, now let us look at air and what how we
use sensors for air in water monitoring the
primary measurement media is air air water
environmental detection, but the variety of
target analytes, it is vast; this is very
very important the variety of target analytes
is vast. So, we are not we are not looking
for 5 targets or 10 targets; we have at least
15 different pesticides equal number of heavy
metals, then we have much much larger number
of antimicrobial resistant genes and then
we have so much nom to measure and then we
have so many other organic materials to measure
so many dyes to measure.
So, the analytes; the domain of analytes is
vast at sites of potential pollution such
as in factory effluent. So, if they have a
factory we know its making 3 kind of dyes
its making four different kinds of pollutants.
So, it will work well for us if you have a
Biosensor that detects the four pollutants
we do not need the one that detects 20, 50,
100 different kinds of targets.
So, it is desirable in ah sites of potential
pollution such as factory affluent way; we
are already expecting pollution potential
position; it is desirable to install online
means data goes to internet directly real
time monitoring an alarm targeted at specific
analytes, but in many 3 cases random or discrete
monitoring or both target species or general
hazardous compound would be sufficient; the
survey of market potential has identified
the increasing significance of this area and
this is now substantiated by a strong interest
from industry.
I want to give an example at the time of recording
the first part of this lecture series ah there
was a very big call from ah dst which is dsd
collaboration with ah partners in the us it
is called ius sdf indo us science and technology
forum and the call was titled research initiative
for real time river water and air quality
monitoring. So, I am highlighting this to
give you an idea that not just ah 3 scientists
not just we environmental engineers and students,
but even our government and the government
of other countries such as us their emphasis.
Now is on how can we monitor data real time
and when we are talking in real time, we do
not want to establish big laboratories right
next to the river, we just want to have a
handheld equipment or a stationary equipment
you install, it you get the data you are fine
whether it is river or air both.
So, this is immense scope of Biosensors here
because Biosensors have the ability to be
very specific and even though enzymes and
antibodies are very sensitive to interference
and temperature changes, we have optimist
again another bio receptor which are very
specific and can be better ah better better
contain their structure as environmental change
environmental changes happen.
Now, another thing; I want to mention is in
the logo of the this particular call it says
internet of things typically when we think
of Biosensors ah we want to get the data maybe
the data is displayed on the screen or maybe
it is collected in a system which I can put
in my computer and get the data, but if I
am interested in real time monitoring it is
very on line real time monitoring, it is very
important that data is shared right as it
is generated with public or with whoever the
intended viewers are in that case we have
something called internet of things.
So, basically you have your Biosensor or any
kind of sensor 
and then it generates data and the data is.
Now let us say some gen generates data the
data is picked up by a mobile phone which
transmits to your big computer in your lab
and then this shows up in on your website.
Now this is what is called internet of things;
I am connecting everything with internet of
things.
Let us say if this shows a warning that hey
something is wrong here then this mobile will
get the information and then the person working
with it will be able to take the right action
already so.
We ah have talked about by a sensor there
is analyte, there is a target. Now what kind
of targets are possible when we talk of Biosensors?
You can be talking up to teens you can be
talking about toxins peptides vitamins sugar
metal and so on and so forth basically now
the list is endless other xenobiotics, we
do not have it here, but we can also detect
pathogens, we can detect antimicrobial resistance
all by using your Biosensors.
Now ah proteins toxins the example proteins
are very simple. Let us say, I want to detect
presence or pry on a particular protein that
I am interested in and then I might people
might create a Biosensor for this toxins;
for example, I want to do environmental analysis
for algal or rather sino toxins, there are
some cyanobacteria some algae that produce
toxins and I want to know whether those toxins
are present or not. Now in order to do that
if I have a handheld I did the handheld monitoring
detection system that will tell me the absent
presence of toxin great peptides hm peptides
could be your genes.
For example the peptides could be other small
ah nuclear peptides on the cell wall that
you can use as detection target then vitamins
sugar a metal iron and. So, on and. So, forth
next a sample hi handling how how do I deliver
the analyte to by receptor. So, here I buy
a receptor let us say its enzyme based . So,
we have enzyme sticking to it now how do I
end this is my analyte; how do I send my analyte
here; that is a very important question.
So, for example, in ph you just dip electrode
into the water right. So, this is important
for example, when you are doing your glucose
measurement you prick the finger and eat the
blood drop you put it on you put it you carry
your finger to the paper strip. So, this is
one of the first question we need to answer
how will the analyte come in contact with
my receptor once you have solved this the
another question comes what if the analyte
is very low in quantity.
For example; only ah only 1 to 10, let us
the only 1 to 10 cells of Kryptos for the
air present . So, how do I detect them because
my my by receptor cannot is not coming in
contact because ah this is too less the concentration
is too less in this case; I can increase the
concentration; however, on the other hand
if I have too much analytes; I might dilute
the sample and then introduce it to my ah
by receptor.
The next phase filtration in selection; I
can select for the contaminant I can filter
the contaminant apart; like for example, I
can filter it because I know cryptosporidium
quite big cell I can filter at 1.545 micron
filter paper and then I will get concentration
of cell and then I can introduce them to my
bio receptor and get a signal .
Now, let us look at some examples of Biosensor;
obviously, the most common is pregnancy test
basically it detects a protein in urine. So,
you have Biosensors here that has past enzyme
that are specific to this particular protein
when when they interact with each other they
give out signal and the signal is a an optical
signal because the colour changes. So, calorimetric
signal and then you can know whether pregnancy
is just passing or failing.
Next is glucose monitoring devices we have
at least two kinds of devices that are popular
in our country right now one is the glucose
strip just prick your finger put a blood drop
here and you get your glucose data here now
again this is your Biosensor here there bio
receptors here that when they interact with
glucose they undergo transform, they undergo
conformational change which is; then read
by this transducer and then output as the
data here next is continuous glucose monitoring
devices.
Now these are very helpful for type one diabetic
patients and what we do is that there are
3 for disposable sensors that are inserted
into the skin and then these sensors interact
with the glucose levels and when glucose falls
below a particular value or interact with
the glucose and whether in glucose falls or
increases there they undergo transformational
change which is picked up by your transducer
and it gives you data here.
Next you have your ah biocore Biosensor platform
sensor typical lab laboratory based platform
and then; obviously, we have over time coal
miners Biosensor. So, I hope you take this
as a joke that for humour that even because
cannery bird this is yellow canary pop by
the way that even the yellow canary bird is
a biological agent of biological entity and
thus taking yellow cannery down into the mind
to test the presence and absence of oxygen
is using a Biosensor already.
And this is our ah one of the leading Biosensors
in the world; it is also being sold in our
country right now it is in infectious disease
Biosensor from RBS and it is really amazing;
there are two different kinds and one is breathalyzer.
So, you can just breathe into it it will collect
your bio aerosols and then because it is a
Biosensor, they will interact with different
bio receptors and then it will give a signal.
So, basically it works this way it will take
sample through you.
Now if you want to test for tuberculosis,
you coughing from one end and when you cough,
it collects the samples and then you place
this in your reader to process and within
2 minutes, it is very rapid it will give tuberculosis
positive or negative I encourage you to go
through their website. So, you can just look
up infectious disease by sensor from RBS and
learn more about what they do how they do
all right.
Now what are typical sensing techniques for
Biosensors typical bout sensing techniques
for Biosensors include fluorescence, DNA microarray,
SPR surface plasmon resonance, impedance spectroscopy,
scanning probe microscopy, AFM, STM, quartz
crystal microbalance SERS; very surface enhanced
Raman micro spectroscopy and then we have
electric or electrochemical Biosensors. Let
us go through them one by one.
In fluorescence based Biosensors, what we
have is we have bio receptors here and in
your bio receptor when you interact with the
analyte when the analyte interacts with the
bio receptor this unit; now this transform
unit; it has the property of fluorescence
which means that if you shine the right wavelength
light on it, it will give you a very measurable
recognized very easily recognizable signal
and and a very good example would be what
we talked about in the previous class where
we had ah cells that release that had undergone
mutation and because of mutation what happens
is that we trigger DNA repair genes.
So, if you have lot of active DNA repair genes,
they will interact with a particular protein
a particular gene in jellyfish. So, this is
called a jellyfish gene and together they
had the property to have fluorescence. So,
if you shine blue laser; what you will get
is green laser and then you know already there
is mutation present potentially cancer present
and then for essence based sensors are also
used when we are looking at different kinds
of assays different kinds of microscopies;
next we have DNA microarray we have talked
about this already, but this is a wonderful
ah opportunity to revisit DNA microarray .
So, this is this this is basically your lab
on chip and you have valves and in each valve
already. So, you have wells on your chip your
DNA microarray chip now in each of these,
you have a particular nucleotide polynucleotide.
Now this poly nucleotide is specific to some
other. So, remember how pairing happens in
DNA, you have for a it will combine with the
T, if you have A G, it wants to combine a
will not bond with C G will not form bond
with T, A will not form bond with G, C will
not form bond with T in if its RNA instead
of T, we could have U.
So, because ATGC ah bonding is very specific,
we can use this as a very specific sensor.
So, if I know the sequence of a particular
agent, for example, I know the sequence of
tuberculosis mycobacteria, then what I can
do is I can find its complementary or at least
for one particular part which is specific
only to mycobacterium which causes tuberculosis
I can make a complementary stand stick it
in here and then if there are tuberculosis
mycobacteria genes present, it will make up
of her complement and when they make a complement,
there is a change in their transferring their
conformation and they can be this can be converted
into an electronic electrical signal and they
need to say yes, there was a mycobacterium
tuberculosis beauty of DNA microarray is that
while this particular well is detecting 4
D vertebral classes, this might be detecting
for gene resistance resistance genes this
might be looking for other kind of bacterial
infections.
So, we can test our samples for thousands
of literally thousands of analytes in one
go. So, that is your DNA microarray then we
have SPR surface plasmon resonance again you
have analyte you have a bio receptor the interact
and then the surface plasmon resonance changes
and when that happens you can detect it convert
it into electronic signal then we have impedance
spectroscopy the very impedance spectroscopy
works is that again you have plate.
And you have your diodes I would make many
of them now each of these diodes act like
receptors they bio receptors and when analyte
sticks with them the impedance which is resistance
to alternating current by the way the impedance
changes. So, because these are ah sorry no
diode these are either cathode or anode and
depending on how your structure is and then
that will change the world that would change
the passing of current.
So, you can have current based impedance meters
or whatever. So, impedance meters. So, what
happens is that you have your bio sense very
receptor here when analyte interacts with
this the impedance of this particular cell
changes and once you know all right even one
analyte was present it changed by x amount
of time when two were present change by 2
into 3 into and so on and so forth; you can
get semi quantitative or quantitative data.
So, this is how impedance spectroscopy works.
Next we have scanning probe microscopy AFM,
STM, basically; what they do is AFM for example,
will tell you the surface information based
on the charges the example if you have a surface
here and you have your ah send bio receptors
here these are bio receptors . So, you have
your bio receptors here. So, if the analyte
comes.
In sticks; whether it undergoes a change and
when it undergoes a change your AFM needle.
So, it is a cantilever with the pin at the
top every needle when it reads the data when
it reaches your surface it will notice oh
there is some change here. So, now, you get
your data. So, this is a SPM scanning probe
microscopy AFM STM based ah Biosensors scanning
tunnelling microscopy also can be used then
you have quartz crystal microbalance works
in similar principle surface enhanced Raman
spectroscopy is a very interesting case. So,
let us look into it.
So, surface enhanced Raman Smotherman microscopy
allows us to detect a very low very small
faint roman signals, but we can enhance them
typically we have enhancing Raman signal is
by using gold. So, if you have; for example,
for example, if you have a Sino toxin and
this work was done at Virginia tech. So, if
you are interested you can look up doctor
peter of excellence work he worked on using
sirs and he is working for on using search
for the detecting different kinds of environmental
ah toxins. So, you have doctor peter of excellent
Peter J Vikesland at virginia tech .
So, he is using sirs quite a leading researcher
and so, if you have a sino toxin and if you
have gold nanoparticles of gold particles
that interact with the sinon toxin when you
shine your laser and when it gives away Raman
signal a very weak, Raman signal your Raman
signal would be enhanced and you will be able
to get very low detection values very high
sensitivity and specificity for your sino
toxin, then we have electrochemical signals
already. Ah
Dear students this is all for today in the
next class, we will go ahead and we look more
about Biosensors, we will try to find out;
what are the different examples of Biosensors
perhaps even go through them the ones that
we talked just now and more and ah we will
also see about what are the applications for
Biosensors where there is scope and perhaps
consider one or two applications. So, that
is all for today.
Thank you .
