BT620 BIOSENSORS

Instructor: Prof. Pranab Goswami

Department: BSBE
Session: July-November 2021
BT 620 BIOSENSORS (3-0-0-6)
Syllabus
Introduction; General configuration of biosensor; Generations of
biosensors; Basic principle and instrumentation of different biosensors:
electrochemical, optical, acoustic, piezoelectric, and calorimetric
biosensors; Biological recognition systems: enzyme, antibody, nucleic
acid, cell, and tissue; Properties of ideal materials for biosensors; Classes
of materials for biosensors: polymers, material containing metal
complex, sol-gel materials, nanomaterials, composite materials, metal
oxides, photonic crystals, and zeolite materials; Application of
biosensors for food and fermentation processes, environment monitoring,
and clinical diagnostics.
Advanced Materials and Techniques for Biosensors and Bioanalytical
Applications, (ed) Pranab Goswami, CRC Press Tailor & Francis, New
York, London (2020)
https://www.amazon.com/Materials-Techniques-
Biosensors-Bioanalytical-
Applications/dp/0367539659

https://www.taylorfrancis.com/books/97810
03083856

https://books.google.co.in/books/about/Advance
d_Materials_and_Techniques_for_Bi.html?id=f3-
JzQEACAAJ&redir_esc=y

https://drukkerijmiddelburg.nl/advanced-
materials-and-techniques-for-biosensors-and-
bioanalytical-applications-9780367539658

https://www.saxo.com/dk/advanced-materials-
and-techniques-for-biosensors-a_pranab-
goswami_hardback_9780367539658
 EVALUATION
Total marks: 100
Quiz 1: 10 : 26th August (Friday)
Quiz II: 10 : 04th November (Friday)
Assignmets: 10 : After midsem
Midsem: 30 : (19-25th September 2022)
Endsem: 40 : (23rd-29th November 2022)
THANKS
INTRODUCTION to BIOSENSORS
 BIOSENSORS

Analytical tool… small (portable), simple (capable of using by semi-skilled operators

or patients themselves), and inexpensive (preferably)

Performance factors

 Selective
 Fast response
 Sensitive (sub-milli molar),
 Accurate (better than +/- 5%)
 Reproducible
 Independent of physical parameters
 Short recovery time
 Functionally Stable (operation and
storage).

 Decentralized testing facility : PoC, home diagnosis, & onsite applications.

 Suitable for remote locations in resource limited environments.
CRITERIA THAT SUPPORT COMMERCIAL SUCCESS
 Multiplex detection facility (simultaneous detection of different analytes from a
single specimen) for PoC applications.
 Non-invasive clinical samples: Saliva, sweats, tears, etc.
 Integration of wireless technology… mobile phone, WiFi, blue tooth, IOT etc

For disease diagnosis in underdeveloped and developing countries:

 WHO suggested ASSURED (Affordable, Sensitive, Specific, User-
friendly, Rapid and Robust, Equipment-free, Deliverable to end-users)
criteria, particularly for PoC applications.
APPLICATIONS
 CLINICAL APPLICATIONS

Potential target / analyte Diagnosis/Analysis

Glucose Diabetes

Albumin, creatinine, protein, urea etc Kidney function

Alk. phosphatase, aspartate transaminase, Liver function

lac.dehydrogenase etc

Calcium, magnesium, phosphorus etc Health of bone

Lipid, cholesterol, HDL, LDL and triglycerides Cardiovascular status

etc

Sodium, potassium, chloride, etc Nerve conduction, muscle

contraction etc

Mutation in gene Genetic disorder & prediction

of various genetic diseases etc
Food & agri products:
Testing tea, coffee, spices (currently electronic nose by using pattern recognition
algorithm methods), residual agrochemicals, pesticides, fungicides, herbicides,
pathogen, toxins, adulteration,

Water quality: toxic metals, compounds, pathogenic organisms, toxins etc.

Detection of plant and animal diseases, Fermentation industry, Military (Biological

warfare, explosive etc).
 Compound annual growth rate

For under developed and developing countries additionally:

Infectious diseases such as, TB, malaria, diarrhea etc.

©2019 Markets and Markets Research Private Ltd.

~200 companies are active in the field of biosensors.
THANKS
General Configuration and Classifications of Biosensors
 General Configuration of Biosensors

Definition
A biosensor is an analytical tool consisting of biologically active material used
in close conjunction with a device that converts a biochemical signal into a
quantifiable electrical signal.
 Classification of Biosensors

Different way of classification:

1. BIORECOGNITION PRINCIPLES:
 Catalytic biosensor &
 Affinity biosensor

2. BIORECOGNITION ELEMENTS:
 Enzyme-Based Biosensors,
 Immunosensors,
 Nucleic Acid/DNA Biosensors,
 Cell-Based Biosensors,
 Biomimetic-Based Biosensors.

3. TRANSDUCER ELEMENTS:
 Calorimetric Biosensors,
 Electrochemical Biosensors,
 Optical biosensors,
 Piezoelectric Biosensors.
 CALORIMETRIC BIOSENSORS

A large number of enzyme-catalyzed reactions are exothermic with a significant

evolution of heat, normally ΔH ~ −10 to −200 kJ mol−1.
The relations among the heat generated, enthalpy change, and temperature change
under adiabatic conditions can be expressed :

Where
Q is the total heat evolved during a catalytic reaction,
ΔH is the molar enthalpy change,
np is the molar number of the product,
ΔT is the temperature change,
Cp is the heat capacity, and
m is the mass of the system in which the reaction takes place.
Conventional mercury-based thermometers are less sensitivity.

More sensitive temperature transducers are:

1) Thermopile (or thermocouple) and
2) Thermistor

Up to 80% of the heat generated in the reaction can be registered

Two major types of thermistors are available, depending upon the decrease or increase in
resistance with increasing temperature.
Negative temperature coefficient (NTC) type
Positive temperature coefficient (PTC) type
Example: A thermopile transducer for the detection of
organophosphate pesticides

Organophosphates include insecticides (malathion, parathion, dichlorvos, etc),

nerve gases (soman, sarin, tabun, VX), Herbicides (tribufos [DEF], merphos)
etc

Organophosphate poisoning can be divided into three broad categories:

(1) muscarinic effects,

(2) nicotinic effects
(3) central nervous system effects.

Organophosphates irreversibly inhibit acetylcholinesterase(AchE)

preventing to break acetylcholine (neurotransmitter).
 Acetylcholine (neurotransmitter)

Acetylcholine is the chief neurotransmitter of the parasympathetic

nervous system, the part of the autonomic nervous system (a branch
of the peripheral nervous system) that contracts smooth muscles,
dilates blood vessels, increases bodily secretions, and slows heart rate
Neurotransmission under normal situation (when no inhibitors/
organophosphates):

• Acetylcholine (ACh) is synthesized from choline and acetyl-CoA through

the action of choline acetyl-transferase in cholinergic neurons.

• When an action potential reaches the terminal button of a presynaptic neuron

a voltage-gated calcium channel is opened, that stimulates the exocytosis of
Ach into the synaptic cleft.

• ACh is released from the nerve into the synaptic cleft and binds to ACh
receptors on the post-synaptic membrane, relaying the signal from the nerve.

• AchE which is located on the post-synaptic membrane, terminates the signal

transmission by hydrolyzing ACh.

• The liberated choline is taken up again by the pre-synaptic nerve and ACh is
synthetized by combining with acetyl-CoA through the action of choline
acetyltransferase.
Acetylcholine formation and function

cholinergic neurons
http://traumagency.blogspot.com/2015/09/organophosphate-poisoning.html
Fig. A type of flow injection calorimetric biosensor. (1) sample container; (2) buffer
container; (3) injection valve; (4) peristaltic pump; (5) thermoelectric thermostat; (6)
enzyme reaction cell; (7) reference cell; (8) thermopile sensor; (9) A/D convertor.
SEE CLASS NOTE
 The sensitivity (10-4 M) and range (10-4 - 10-2 M) of calorimetric biosensors are
low.
 The sensitivity can be increased by increasing heat output by the coupled reaction
steps all of which contribute to the heat output.
Example: The sensitivity of the glucose sensor using glucose oxidase can be more than
doubled by the co-immobilization of catalase within the column reactor in order to
disproportionate the hydrogen peroxide produced.

In spite of the +Ve enthalpy of the pruvate kinase reaction, the

overall process results in a 1000 fold increase in sensitivity
  Such reaction systems do, however, have the serious disadvantage in that they increase
the probability of the occurrence of interference in the determination of the analyte of
interest.
 The advantages of the calorimetric biosensor are its general applicability and the
possibility for its use on turbid or strongly coloured solutions.
 The most important disadvantage is the difficulty in ensuring that the temperature
of the sample stream remains constant (± 0.01°C).

Commercially available device: Of the thermal biosensors only the enzyme thermistor is
commercially available. Example: Thermal assay probe developed by ThermoMetric Co.,
Sweden.

******************
PIEZOELECTRIC BIOSENSORS
Greek piezo means to squeeze or press.

A material that generates an electric charge when mechanically deformed.

Conversely, when an external electric field is applied to piezoelectric materials they
mechanically deform.

Fig. A piezoelectric disk generates a voltage when deformed

The piezoelectric effect is used to measure changes in pressure,
acceleration, temperature, strain, or force by converting them to an
electrical charge.

Many materials, both natural and man-made, exhibit piezoelectricity

Example:
Naturally-occurring crystals: berlinite (AlPO4, structurally identical to
quartz), cane sugar, quartz, Rochelle salt, topaz, Dry bone, Tendon, Silk
, Wood, Enamel etc.
Man-made crystals: gallium orthophosphate (GaPO4), Langasite
(La3Ga5SiO14), Man-made ceramics, Polyvinylidene fluoride
Mechanism:

The piezoelectric effect is closely related to the occurrence of electric

dipole moments (P) in solids.
The P may either be induced for ions on crystal lattice sites with
asymmetric charge surroundings or may directly be carried by
molecular groups (as in cane sugar).
Dipole moment can be defined as the product of magnitude of charge
and the distance of separation between the charges (C.m).
In the simple case of two point charges, one with charge +q and one with
charge −q, the electric dipole moment p is:
P = qd
Where d is the displacement vector pointing from the negative charge to
the positive charge.

The dipole moment of an array of charges,

Piezoelectricity is caused by dipole density [Cm/m3] ) (Cm is coulomb-
meter) in the bulk.

Piezoelectric materials also show the opposite effect, called converse piezoelectric
effect, where the application of an electrical field creates mechanical deformation in the
crystal.

Thus an anisotropic crystals (crystals without a center of symmetry) can generate an

electric dipole when mechanically squeezed.
The effect can also work in an opposite way in that an anisotropic crystal deformed
(oscillate) when voltage is applied on it.

Analyte can be determined from the electricity produced on the crystal surface
through interaction with either crystal alone or electrode.
Principle of operation
The way a piezoelectric material is cut produces three main operational
modes:

(a)Transverse effect: A force applied along a neutral axis (y) displaces

charges along the (x) direction, perpendicular to the line of force. The
amount of charge (C x) depends on the geometrical dimensions of the
respective piezoelectric element. When dimensions a , b , c apply,
C x = d xy F y b / a
where a is the dimension in line with the neutral axis, b is in line with
the charge generating axis and d is the corresponding piezoelectric
coefficient.
(b) Longitudinal effect: The amount of charge displaced is strictly
proportional to the applied force and independent of the piezoelectric
element size and shape. Putting several elements mechanically in series
and electrically in parallel is the only way to increase the charge output.
The resulting charge is
Cx = d xxF xn
where d x x is the piezoelectric coefficient for a charge in x-direction
released by forces applied along x-direction. Fx is the applied Force in x-
direction and n corresponds to the number of stacked elements
( c) Shear effect: The charges produced are strictly proportional to the
applied forces and independent of the element size and shape. For n
elements mechanically in series and electrically in parallel the charge is
Cx = 2 d xx F x n

In contrast to the longitudinal and shear effects, the transverse effect makes it
possible to fine-tune sensitivity on the applied force and element dimension.
The bound mass on the crystal surface causes slowing of oscillation.
For the common quartz crystals, the frequency shift (Δf) is directly proportional to the
change of mass (Δm) on the crystal, as described by Sauerbrey:

f0 is the fundamental mode of the crystal oscillation (in hertz),

A is the piezoelectrically active area (in centimeters),
ρq means density (2.648 g/cm3), and
μq means shear modulus (2.947 × 1011 g/cm·s2) of quartz.
The Sauerbrey equation is reliable for calculating frequency shift when the ambient
environment is not unaltered.
Upon a change in the surrounding environment, since viscosity has an impact on
frequency shift, the equation described by Kanazawa and coworkers for quartz crystal
should be consulted.

The equation states that frequency shift is proportional to an increase of

ambient viscosity η (the symbols with index l relate to the ambient liquid
and q to quartz crystal).
 Frequency response of a
piezoelectric sensor;
output voltage vs applied force

Piezoelectric biosensors can be of two different types:

 Bulk acoustic wave (BAW) and Surface acoustic wave (SAW) piezoelectric
sensors.
 In BAW the wave propagates through the interior of the substrate.
 In SAW the wave propagates on the surface of the substrate.

These sensors apply an electric field that creates mechanical stress (wave), which
moves through/on the substrate, and in the last step, is converted back to an electric
field before we can measure it.

As the wave propagates through/on the surface of the substrate, its velocity continually
changes (as kown by measuring the change in the frequency). This can be related to
the physical mass being measured.
A quartz crystal microbalance (QCM) is one of the simplest BAW devices.

 This piezoelectric technique is known for its excellent sensitivity, and hence it has
wide applications in the medical, aeronautical, and telecommunications, fields.
 These are suitable for label-free and real-time biosensing.
 They can attained detection limits to the pico level and hence are suitable to
measure various gases such as ammonia, hydrogen, methane, and carbon
monoxide.
 These sensors have good compatibility with integrated circuits (IC)
technology and can be easily manufactured by photolithography, which
renders them inexpensive.
• A QCM-based biosensor was constructed for the determination of
organophosphorus and carbamate pesticides in the nM level.
• The sensor had an immobilized enzyme acetylcholinesterase, which
converted 3-indolyl-acetate to insoluble indigo pigment, providing
alteration in the oscillations.
• The biosensor was used for the assay of pesticides, which inhibit the
enzyme acetylcholinesterase.
• When the enzyme became inhibited, the precipitate was not formed,
hence no change of mass observed.
Example: A rapid method for the diagnosis of tuberculosis and other
infections caused by mycobacteria

Almost all current methods of diagnosing TB have drawbacks:

• They tend to be either nonspecific or too time-consuming.

• In most cases of pulmonary and extrapulmonary TB, diagnosis

depends upon culturing the mycobacterial organism, a process
requiring 4-8 weeks.

• some of them do not have the high specificity or sensitivity required

for proper diagnosis
Materials and fabrication:

•Here construction of antibody-based piezoelectric crystals capable of

detecting mycobacterial (M. tuberculosis) antigens.
•The antigen here is a secretory protein and potent T cell antigen.
•Detector crystal is alpha quartz crystals.
•These crystals are insoluble in water and resistant to high temperatures
up to 579°C with no loss of piezoelectric properties.
•The crystals were 10 MHz AT-cut quartz crystals with an electrode
coating deposited on each side using sputtering method.
•The optimum thickness of the gold electrode layer was estimated at
1,000 Angstroms.
•The crystal was mounted on a holder with stainless steel with leads.
•A silver composite was used to connect the electrode to wire.
•The crystals were 14 mm in diameter, and the electrodes on both sides
of the crystal were 8 mm in diameter.
Experimental apparatus for the piezoelectric sensor
• The crystal is driven by a low-frequency transistor oscillator (1-30 V
power supply) and set at 9 V.
• The frequency of the vibrating crystal was monitored by a frequency
counter.
• The crystal electrodes were first modified with a coating of protein A
for better adhesion of the antibodies to the surface of the transducer.
• Protein A is a polypeptide isolated from Staphylococcus aureus that
binds specifically to the IgG antibodies, without interacting at the
antigen site.
• This property permits the formation of tertiary complexes consisting
of protein A, antibody, and antigen.
• After each step in the coating process-first with the metal depositions
and then with the biomolecular analytes-the frequency reading was
recorded.
• The crystals were methodically dried and following each step the
frequency of the crystal was recorded.
• The difference in frequency change between the control and
experimental crystals were compared.
THANKS
ELECTROCHEMICAL BIOSENSORS

 Amperometric Biosensors
 Potentiometric Biosensors
 Conductometric Biosensors
AMPEROMETRIC BIOSENSORS

2
O2 as redox entity H2O2 as redox entity
What is volt?
 volt is simply the energy (J) required to move charge (c).
 Reactions are initiated by applying a potential,
V = Joule/Coulomb
 Application of a voltage to an electrode therefore supplies
electrical energy.
 Since electrons possess charge an applied voltage can alter the
'energy' of the electrons within a metal electrode.
Which electron in a metal?
•Electrons of the Fermi-level (EF).
•The Fermi-level corresponds to the energy of the highest occupied
orbitals.
•This level is not fixed and can be moved by supplying electrical energy.
•Electrochemist's are therefore able to alter the energy of the Fermi-level
by applying a voltage to an electrode.
•Depending upon the position of the Fermi level it may be
thermodynamically feasible to reduce/oxidise a species in solution.
The figure below shows the Fermi-level within a metal along with the orbital
energies (HOMO and LUMO) of a molecule (O) in solution.

Voltage • Left: EF < the E of LUMO of (O), hence thermodynamically

unfavourable for an e to jump from the electrode to O.
• Right: EF > the E of LUMO of (O), it is thermodynamically favourable
for the electron transfer to occur to O.
The fundamental relationship between the current flowing and the applied voltage is given by
the following Butler-Volmer equation,.

This expression shows how the current will respond to changes in potential, the value of alpha
and the quantity io which is called the exchange current (density). The term alpha reflects the
sensitivity of the transition state to the applied voltage. If alpha = 0 then the transition state
shows no potential dependence. Typically alpha = 0.5 this means that the transition state
responds to potential in a manner half way between the reactants and the products response.

Here we wish to see how the voltage influences the current in the absence of concentration
effects. To do this we will assume that the a solution is well mixed ie that the surface and bulk
concentrations are identical which will be reasonable under condition of small current flow.
Now the Butler-Volmer equation simplifies to

Without concentration
Without concentration and
and therefore
therefore mass
mass transport
transport effects
effects to
to complicate
complicate the
the electrolysis
electrolysis it
it
is possible
is possible toto establish
establish the
the effects
effects of
of voltage
voltage on
on the
the current
current flowing.
flowing. InIn this
this situation
situation the
the quantity
quantity
E -- E
E Eee reflects
reflects the
the activation
activation energy
energy required
required to
to force
force current
current ii to
to flow.
flow.
• Amperometry is a subclass of voltammetry in which the electrode is held at
constant potentials for various lengths of time.
• Voltammetry applies a constant and/or varying potential at an electrode's surface
and measures the resulting current with a three electrode system.
• To conduct such an experiment requires at least two electrodes.

Three-electrode setup:
(1) working electrode;
(2) auxiliary electrode;
(3) reference electrode
Working electrode: It makes contact with the analyte, must apply the desired potential
in a controlled way and facilitate the transfer of electrons to and from the analyte.

Reference electrode: It acts as the other half of the cell, has a known potential with
which to gauge the potential of the working electrode.
It may use to balance the electrons added or removed by the working electrode. While
this is a viable setup, it is extremely difficult for an electrode to maintain a constant
potential while passing current to counter redox events at the working electrode. To
solve this problem, the role of supplying electrons and referencing potential has been
divided between two separate electrodes.

Aauxiliary electrode: It passes all the current needed to balance the current observed at
the working electrode. To achieve this current, the auxiliary will often swing to extreme
potentials at the edges of the solvent window, where it oxidizes or reduces the solvent
or supporting electrolyte.

These electrodes, the working, reference, and auxiliary make up the modern three
electrode system.
Fast
Sensitive
Selective
Label free
Min. or no sample loss
Small size-low cost