NPTEL – Nanotechnology - Nanobiotechnology
Capture-based, Cell-based and Tissue-based
sensors
K. Uma Maheswari
Professor, School of Chemical & Biotechnology
SASTRA University
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Table of Contents
Preface ........................................................................................................................... 3
1 What are biosensors? ................................................................................................ 3
2 Major requisites of an ideal biosensor – The 7 ‘S’ rule! .......................................... 4
3 Classification of biosensors ...................................................................................... 4
3.1 Qualitative and Quantitative sensors ………………...………………………………4
3.2 External detection, Final detection & In vivo detection devices……………………5
3.3 Classification based on bioreceptors…………..……………………………………..5
4 Capture-based sensors.............................................................................................. 6
4.1 Antigen-Antibody interactions………………………………….………………………6
4.1.1 Direct Method………………..……………………………………………………7
4.1.2 Indirect Method………………..………………………………………………….7
4.2 ELISA……………………………………………………………………….……………8
4.3 Radioimmune assay…………………………………………………………………….9
4.4 Enzyme - Substrate Interactions……………………………………………..………10
5 Reference .................................................................................................................. 11
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Module Objective
At the end of the module, the learner would be familiar with the different types of
biosensors, the sensing mechanisms involved and the salient features of the sensors.
Preface
“If you can’t measure something, you can’t understand it. If you can’t understand it, you
can’t control it. If you can’t control it, you can’t improve it”, said the American author,
engineer and entrepreneur H. James Harrington. Sensors are devices that enable detection
and quantification of a specific molecule. Sensors have been widely used in food quality
control, pollution monitoring, defense applications, clinical diagnostics and forensics.
The integration of biology, electronics, chemistry, physics and nanotechnology has led to
the evolution of a novel breed of nanobiosensors that havesuperior performance when
compared with conventional sensors. The following lectures provide an insight into the
different classes of biosensors and their mechanisms of sensing.
This lecture provides an overview of biosensors and capture-based sensors.
1 What are biosensors?
A biosensor is an analytical device consisting of two important components namely a
bioreceptor that serves as a recognition site for the analyte(sensing element) and the
transducer (transducing element), which converts the response into electrical signals.
When the transducers are based on the integrated circuit microchips, it is often referred as
‘biochips’. Biosensors have been widely used in three broad areas namely biological
monitoring (clinical diagnosis), food quality monitoring and environmental sensing
(pollution monitoring).
The bioreceptors are the heart of a biosensor. These are based on biomolecules that
exhibit specific and selective interactions with an analyte, which results in a change in the
property of the biomolecule that is in turn detected and measured by the transducer. The
time taken for the property change in the biomolecule on interaction with the analyte is
extremely small thus giving a very fast response time. These properties are invaluable in
sensing applications thus making the biosensors much superior to the conventional
chemical sensors. The most commonly employed bioreceptors are antibodies, enzymes
and oligonucleotides.
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2 Major requisites of an ideal biosensor – The 7 ‘S’ rule!
The development of a biosensor is driven by the need to achieve the following main
requirements.
• Specificity: The sensing element should exhibit response only to the analyte even
in the presence of numerous molecules that may resemble the structure or
function of the molecule of interest
• Sensitivity: The sensor should be able to detect extremely low levels of the
analyte. Currently, super ultrasensitive nanobiosensors withcapability to sense
even in zeptomolar (10-21 M) concentrations have been reported!
• Speed: The sensor should be able to produce a signal in response to the analytein
the shortest possible time. In other words, it should possess very short response
times. If the sensor has to be used for another cycle of measurements, then it
should be able to return to its base value (original condition) quickly. In other
words, the sensor should possess a very short recovery time
• Stability: The sensing element should exhibit both structural and functional
stability in order to be able to perform as a sensor
• Small analyte volume:An ideal sensor should be able to detect the analyte even
in µL and sub- µL volumes
• Small size: Portability is an added incentive in a sensor so that it can be used
anywhere
• $:A very important criterion in any commercial sensor is to be cost-effective,
thereby saving $$$!
3 Classification of biosensors
3.1 Qualitative and quantitative sensors
Biosensors can be categorized as qualitative and quantitative sensors depending on
whether they detect the presence of the analyte in the sample (qualitative) or they also
provide information on the amount of the analyte present in the sample (quantitative).
The urine test strips available commercially is an example of a qualitative sensor that
gives information about the presence of glucose, ketones, bilirubin, protein and
leukocytes in urine while the blood glucose sensor used in biochemical labs is a
quantitative sensor that provides information on the amount of glucose present in the
blood sample.
3.2 External detection, field detection & In vivo detection devices
Based on the detection devices, biosensors can be classified as external detection, field
detection and in vivo detection devices. An external detection device usually involves the
use of large instruments for detection with maximum sensitivity and high throughput.
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SQUID (Super conducting quantum interference devices) sensors are an example of this
class of sensors.
Field detection device on the other hand needs to be simpler, portable and is more robust.
The hand-held glucose sensors that are available in the market are classic examples of
field detection devices.
In in vivo detection devices, the sensing element in the form of a small catheter probe is
introduced inside the body, which are minimally invasive. The sensor response is then
detected using suitable detectorsthat are usually kept outside the body.This type of
system is limited by biofouling that typically limits the lifetime of in vivo measurements
to 1-2 days. What is biofouling? When a probe is inserted in the biological system, there
can be formation of a biofilm due to bacterial adhesion and multiplication. The nature of
the biofilm as well as the thickness of the film formed depends on the location of the
sensor probe. The bacterial film can cause secondary infections if fragments of the film
travel to remote locations in the body. Other major problems involved in in vivo detection
include the activation of the immune response of the biological system against the foreign
material (the probe, in this case). This activation is usually manifested in the form of
inflammation, fibrosis and loss of vasculature due to insertion of the probe.
3.3 Classification based on bioreceptors
Biosensors can also be classified into different types based on the bioreceptors (sensing
element) Bioreceptor is basically a biological component, which may be an antibody,
enzyme, protein, nucleic acid or even whole cells that involves a biochemical
transformation for the recognition process. Thus these bioreceptors form the sensing
element in a biosensor. Based on the type of biological entity employed for sensing,
biosensors can be categorized as:
• Capture-based sensors
• Cell-based sensors
• Tissue-based sensors
Capture-based sensors are based on the principle of specific binding interactions between
the bioreceptor and the analyte molecule. The capture-based sensors involve interactions
involving an antigen and antibody, complementary strands of oligonucleotides or
polynucleotides, enzyme and its substrate, receptor and its ligand and more recently
interactions using biomimetic molecules. Most of the biosensors developed currently are
capture-based sensors. The capture-based sensors are highly specific.
A cell-based sensor utilizes a single cell (microbial or mammalian) for sensing. The
advantage is that the cell signaling machinery amplifies the signal obtained from a cell in
response to an analyte and hence the sensitivity is very high.Sensitivity represents the
lowest concentration of an analyte that can be detected by the sensor.
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The tissue-based
based sensors use a mass of cells for the sensing process. The advantage of
such systems is their ability to use the amplification network of the cells in the tissue
thereby producing a measurable signal even for extremely low quantities of the analyte.
The sensitivity of such systems will therefore be very high.Let Let us look at these
interactions
tions and their implications on the sensing property in the following sections.
section
4 Capture-based
based sensors
The capture-based
based sensors comprise the largest class of sensors. The most common
interactions that have been explored for development of the sensing element
e are
discussed in the following sections.
4.1 Antigen-Antibody
Antibody Interactions
The binding affinity and specificity between an antigen and its corresponding antibody is
perhaps unmatched by any other kind of interactions. Sensors employing such antigen-
antibody interactions are known as immunosensors and have been extensively
investigated for diagnosis of many clinical disorders. The basis of this sensor is the
highly specific binding of the antigen to its antibody.. Each antigen can have a unique
antibodyy just like each lock has a specific key! For the sensing application, it is essential
that the antigen or antibody
ibody is immobilized on a solid inert matrix. The chemistry used
for the immobilization has a key role to play in determining the sensing efficiency
efficien of the
device. Caution has to be exercised in ensuring that the immobilization technique does
not affect the binding site of the antibody/antigen. The general schematic of an
immunosensor is shown in Figure 1.
Fig. 1: Schematic representation of an immunosensor
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The detection can be carried out either using:
• Direct method
• Indirect method
4.1.1 Direct method
In the direct method, the antigen or antibody will be immobilized on a solid support. On
addition of the sample,the corresponding antibody/antigen from the sample will bind to
its respective immobilized target. The unreacted sample will be washed away. Ifthe
analyteis fluorescent,its emission intensity upon binding with the immobilized target
molecule can be recorded as the signal. The intensity of the emission can be directly
correlated with the concentration of the analyte in the sample.
4.1.2 Indirect method
If the analyte molecule is non-fluorescent, then a two-step reaction involving a secondary
antibody conjugated to a fluorophore (fluorescent molecule)is employed. This method is
referred as the ‘indirect method’. The initial step involves the binding of the antigen with
its corresponding antibody, which is referred as the ‘primary antibody’. After allowing
sufficient time for the antigen-antibody interactions, the unbound reagents are washed
away. The secondary antibody conjugated with a fluorophoreis added to the system after
removal of the unreacted sample. Then, the excess antibody is washed off. The nature of
the secondary antibody is such that it will exhibit specificity to the bound antibody
(which is now referred to as the primary antibody). The unbound antibodies are removed
by washing.The binding of the fluorophore tagged secondary antibody will result in
fluorescent emission. The intensity of the emission is directly proportional to the amount
of analyte present. Instead of a fluorophore, a dye molecule may also be employed for
quantification. Such immunosensors have been extensively used for clinical diagnosis.
Let us look at some examples of immunosensors that are well known in the medical field.
The ELISA assay for detecting viral infections is a popular immunosensor. The principle
of immunosensing has also been employed in the point-of-care (PoC) sensors like the
pregnancy sensor, which detects the hCG (human chorionic gonadotropin) levels in the
urine. Immunosensors for detection of prostate specific antigen (prostate cancer marker),
neuron specific enolase (lung cancer marker), myoglobin (muscle injury and ischemic
marker), cholera toxin (marker for Vibrio cholerae infections) and fatty acid binding
proteins (lipid metabolism markers) are also in different stages of commercialization.
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4.2 ELISA
ELISA (Enzyme-linked immunosorbentassay) is a widely practiced diagnostic technique
that is based on antibody-antigen interactions. This technique employs the same steps as
described in the indirect method except for the fact that the fluorophore-tagged secondary
antibody is replaced by an enzyme-tagged secondary antibody (and hence the name
enzyme-linked immunosorbent assay). After binding of the secondary antibody, the
substrate for the enzyme is added which gets converted to a coloured product whose
intensity is quantified as a measure of the analyte concentration. Generally, the enzyme
employed in ELISA is horse radish peroxidase which can convert hydrogen peroxide to
water and oxygen radicals. The oxygen oxidizes a dye tetramethylbenzidene (TMB),
which turns blue on oxidation due to the reaction shown in Figure 2.
Fig. 2: Conversion of tetramethylbenzidene (TMB) from reduced to oxidized form in the presence of hydrogen
peroxide (H2O2) and horseradish peroxidase (HRP)
Quantification of the intensity of the blue colour developed gives a measure of the
analyte present in the sample. The ELISA technique has been used for detecting HIV
(human immuno-deficiency virus) and HBV (hepatitis B virus) infections. The principle
of ELISA is depicted in the animation given in Figure 3.
Fig. 3: Principle of ELISA
Note; Can be viewed only in Acrobat Reader 9.0 and above
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4.3 Radioimmunoassay
Yet another strategy that has been used to quantify the antigen-antibody interactionsis
based on competitive binding between a fluorescently labeled and unlabelled antigen for
a specific binding site. A known quantity of fluorescently labeled antigen is added to a
system containing its antibody. On addition of the sample containing an unknown
quantity of the same antigen, competitive binding of the unlabeled antigen from the
sample with the antibody occurs resulting in displacement of the labeled antigen. Why is
the labeled antigen displaced? The displacement is driven by the higher concentration of
the unlabeled antigen from the sample! If the fluorescent label is replaced with a radio-
labeled antigen (antigen containing a radioactive isotope), then the technique is known as
radioimmunoassay (RIA).
Did you know?...
The radioimmunoassay technique was first developed by Prof. Rosalyn Yalow and
Prof. Solomon Aaron Berson from the Mount Sinai School of Medicine. Their path-
breaking work on quantifying human insulin using RIA won Prof. Yalow the Nobel
Prize for Medicine in the year 1977. Interestingly, both scientists did not patent this
assay as they felt that this technique should be available freely to everyone thereby
paving way for identification of many endocrinological disorders!
Generally, the radiolabeling is carried out by introducing gamma-emitting radioisotopes
of iodine to a tyrosine moiety in the antigen. This technique finds extensive applications
especially in pharmacology, clinical chemistry, forensic science, environmental
monitoring, molecular epidemiology and agricultural science. However due to the limited
shelf life of radioisotopes, it has been widely replaced by ELISA. The cost of the
radioimmunoassay is less when compared with other immune complex detecting methods
and the sensitivity of this method is very high. Hence, in the recent years, RIA is slowly
regaining its popularity in clinical diagnosis. The principle of RIA is pictorially depicted
in Figure 4.
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Fig. 4: Principle of Radioimmunoassay
4.4 Enzyme-substrate
substrate interactions
After the antigen-antibody
antibody interactions, eenzymes are the next popular choice for
biosensor applications.s. Enzymes have been employed both for their specificity and their
catalytic activity. Many enzymes do not require any co co-factors or co-enzymes
enzymes for their
catalytic activity. But several enzymes can only be catalytically active only in the
presence of inorganic ions like Mg2+, Mn2+ or Zn2+(co-factors)
factors) ororganic moieties such as
+
NADP (nicotinamide adenine dinucleotide phosphate), FAD (flavine adenine
dinucleotide), which are known as coenzymes
coenzymes. The enzymatic catalysis results in a redox
reaction that causes transfer of electrons leading to changes in the current flowing
through the system. This current flow can be measured to quantify the amount of
substrate present in the sample. The glucose sensor that is widely used in biochemical
labs is a typical
ical example of a biosensor based on the enzyme-substrate
substrate interactions.
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Another mode of quantification employing enzyme-substrate interactions in capture-
based sensors involves determination of the pH changes accompanying the catalytic
reaction. For example, the hydrolysis of the substrates penicillin and ampicillin by the
enzymes penicillinase and ampicillinase respectively result in a reduction in the pH,
which can be quantified. Yet another biosensor based on the same principle and same
detection mechanism is the glucose sensor where, glucose oxidase enzyme converts
glucose to gluconic acid causing a reduction in the pH. Figure 5 shows the enzyme-
catalyzed conversion of glucose to gluconic acid.
Fig. 5: Enzymatic conversion of glucose to gluconic acid
The commercial glucose monitor employs glucose oxidase immobilized strips which
when in contact with the sample catalyses the conversion of glucose to gluconic acid.
This process involves transfer of electrons that alters the current as well as redox
potential of the system. This change is measured and correlated with the glucose content
in the sample.
5 Reference
Biosensors and biochips: advances in biological and medical diagnosis,Tuan vo-Dinh,
Drian Cullum, Freselaus J Analytical Chemistry, 2000, 366, 540-551.
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