UNIT - 3

Biorecognition systems:
Enzymes; Oligonucleotides and Nucleic Acids; Lipids
(Langmuir-Blodgett bilayers, Phospholipids, Liposomes);
Membrane receptors and transporters; Tissue and
organelles (animal and plant tissue); Cell culture;
Immunoreceptors; Chemoreceptors; Limitations &
problems. Immobilization of biomolecules.

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 1st Components: Bioreceptors
 In a biosensor, the bioreceptor is designed to interact with the
specific analyte of interest to produce an effect measurable by the
transducer.
 High selectivity for the analyte among a matrix of other chemical
or biological components is a key requirement of the bioreceptor.
 While the type of biomolecule used can vary widely, biosensors
can be classified according to common types of bioreceptor
interactions involving: antibody/antigen, enzymes/ligands, nucleic
acids/DNA, cellular structures/cells, or biomimetic materials.

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 Biorecognition systems : Antibody/antigen interactions
 An immunosensor utilizes the very specific binding affinity
of antibodies for a specific compound or antigen.

 The specific nature of the antibody-antigen interaction is

analogous to a lock and key fit in that the antigen will only bind
to the antibody if it has the correct conformation.

 Binding events result in a physicochemical change that in

combination with a tracer, such as a fluorescent molecules,
enzymes, or radioisotopes, can generate a signal.
Bioreceptors

 Antibody Antibodies are biological molecules that exhibit

very specific binding capabilities for specific
structure (antigens).

membrane

Antigen It can be recognized by antibody.

 Bioreceptor

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Limitations:
There are limitations with using antibodies in sensors:

1. The antibody binding capacity is strongly dependent on assay

conditions (e.g. pH and temperature) and

2. The antibody-antigen interaction is generally irreversible.

However, it has been shown that binding can be disrupted

by chaotropic reagents, organic solvents, or even ultrasonic radiation.
 Biorecognition systems : Artificial binding proteins
The use of antibodies as the bio-recognition component of biosensors has
several drawbacks.
They have high molecular weights and limited stability, contain essential
disulfide bonds and are expensive to produce.
In one approach to overcome these limitations, recombinant binding
fragments (Fab, Fv or scFv) or domains (VH, VHH) of antibodies have
been engineered.
In another approach, small protein scaffolds with favorable biophysical
properties have been engineered to generate artificial families of Antigen
Binding Proteins (AgBP), capable of specific binding to different target
proteins while retaining the favorable properties of the parent molecule.
The artificial binding proteins are much
smaller than antibodies (usually less
than 100 amino-acid residues), have a
strong stability, lack disulfide bonds and
can be expressed in high yield in
reducing cellular environments like the
bacterial cytoplasm, contrary to
antibodies and their derivatives.
They are thus especially suitable to
create biosensors.
 Biorecognition systems : Enzymatic interactions
Enzymes are macromolecular biological catalysts. Enzymes accelerate chemical reactions. The molecules upon
which enzymes may act are called substrates and the enzyme converts the substrates into different molecules known
as products. Metabolic pathways depend upon enzymes to catalyze individual steps. The study of enzymes is called
enzymology. Enzymes are known to catalyze more than 5,000 biochemical reaction types. Most enzymes are
proteins, although a few are catalytic RNA molecules. The latter are called ribozymes. Enzymes' specificity comes
from their unique three-dimensional structures.

Like all catalysts, enzymes increase the reaction rate by lowering its activation energy. Some enzymes can make
their conversion of substrate to product occur many millions of times faster. An extreme example is orotidine 5'-
phosphate decarboxylase, which allows a reaction that would otherwise take millions of years to occur in
milliseconds. Chemically, enzymes are like any catalyst and are not consumed in chemical reactions, nor do they
alter the equilibrium of a reaction. Enzymes differ from most other catalysts by being much more specific. Enzyme
activity can be affected by other molecules: inhibitors are molecules that decrease enzyme activity, and activators
are molecules that increase activity. Many therapeutic drugs and poisons are enzyme inhibitors. An enzyme's
activity decreases markedly outside its optimal temperature and pH.

Some enzymes are used commercially, for example, in the synthesis of antibiotics. Some household products use
enzymes to speed up chemical reactions: enzymes in biological washing powders break down protein, starch or fat
stains on clothes, and enzymes in meat tenderizer break down proteins into smaller molecules, making the meat
easier to chew.
 Biorecognition systems : Enzymatic interactions
The specific binding capabilities and catalytic activity of enzymes make
them popular bioreceptors. Analyte recognition is enabled through several
possible mechanisms:

1) the enzyme converting the analyte into a product that is sensor-

detectable,

2) detecting enzyme inhibition or activation by the analyte, or

3) monitoring modification of enzyme properties resulting from interaction

with the analyte.

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 Bioreceptor

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The main reasons for the common use of enzymes in biosensors are:

1) Ability to catalyze a large number of reactions;

2) Potential to detect a group of analytes (substrates, products,
inhibitors, and modulators of the catalytic activity); and
3) Suitability with several different transduction methods for
detecting the analyte.

• Notably, since enzymes are not consumed in reactions, the

biosensor can easily be used continuously.
• The catalytic activity of enzymes also allows lower limits of
detection compared to common binding techniques.
• However, the sensor's lifetime is limited by the stability of the
enzyme.
 Biorecognition systems : Affinity binding receptors
A receptor is a protein which binds to a specific molecule. The molecule it binds is
known as the ligand. A ligand may be any molecule, from inorganic minerals to
organism-created proteins, hormones, and neurotransmitters. The ligand binds to the
ligand-binding site on the receptor protein. When this binding happens, the receptor
undergoes a conformational change. This change is shape slightly alters the protein’s
function. From this, a number of things can happen. The conformational change in the
receptor can cause the receptor to become an enzyme and actively combine or separate
certain molecules.

The change can also cause a series of changes in related proteins, eventually
transferring some sort of message to the cell. This message could be a metabolic
regulation message, or it could be a sensory signal. The receptor has a certain capacity
to hold onto the ligand, known as the binding affinity. Once this attraction wears out,
the receptor will release the ligand, undergo a change to the original shape, and the
message or signal will end. The speed of this turnover depends on the strength of the
affinity between receptor and ligand.
 Biorecognition systems : Affinity binding receptors
Antibodies have a high binding constant in excess of 10^8 L/mol, which
stands for a nearly irreversible association once the antigen-antibody
couple has formed.

For certain analyte molecules like glucose affinity binding proteins exist
that bind their ligand with a high specificity like an antibody, but with a
much smaller binding constant on the order of 10^2 to 10^4 L/mol.

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 Biorecognition systems : Nucleic acid interactions
 Nucleic acids are polynucleotides—that is, long chainlike molecules composed of a series of
nearly identical building blocks called nucleotides. Each nucleotide consists of a nitrogen-
containing aromatic base attached to a pentose (five-carbon) sugar, which is in turn attached to a
phosphate group. Each nucleic acid contains four of five possible nitrogen-containing bases:
adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). A and G are categorized as
purines, and C, T, and U are collectively called pyrimidines. All nucleic acids contain the bases
A, C, and G; T, however, is found only in DNA, while U is found in RNA.

 The pentose sugar in DNA (2′-deoxyribose) differs from the sugar in RNA (ribose) by the
absence of a hydroxyl group (−OH) on the 2′ carbon of the sugar ring. Without an attached
phosphate group, the sugar attached to one of the bases is known as a nucleoside. The
phosphate group connects successive sugar residues by bridging the 5′-hydroxyl group on one
sugar to the 3′-hydroxyl group of the next sugar in the chain. These nucleoside linkages are
called phosphodiester bonds and are the same in RNA and DNA.

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 Biorecognition systems : Nucleic acid interactions
 DNA is a polymer of the four nucleotides A, C, G,
and T, which are joined through a backbone of
alternating phosphate and deoxyribose sugar
residues. These nitrogen-containing bases occur in
complementary pairs as determined by their
ability to form hydrogen bonds between them. A
always pairs with T through two hydrogen bonds,
and G always pairs with C through three hydrogen
bonds. The spans of A:T and G:C hydrogen-
bonded pairs are nearly identical, allowing them
to bridge the sugar-phosphate chains uniformly.
This structure, along with the molecule’s chemical
stability, makes DNA the ideal genetic material.
The bonding between complementary bases also
provides a mechanism for the replication of DNA
and the transmission of genetic information. Figure: DNA structure
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 Biorecognition systems : Nucleic acid interactions
 Biosensors that employ nucleic acid interactions can be referred to
as “Genosensors”.
 The recognition process is based on the principle of
complementary base pairing, adenine:thymine and cytosine:guanine
in DNA.
 If the target nucleic acid sequence is known, complementary
sequences can be synthesized, labeled, and then immobilized on the
sensor.
 The hybridization probes can then base pair with the target
sequences, generating an optical signal.
 The favored transduction principle employed in this type of sensor
has been optical detection.
 DNA structure
Another biorecognition mechanism
involves hybridization of
deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA), which are the
building blocks of genetics.

Four chemical bases:

• adenine(A), guanine (G),
• cytosine (C), and thymine (T)
 Oligonucleotides
• Oligonucleotides are short DNA or RNA molecules, oligomers, that
have a wide range of applications in genetic testing, research, and
forensics.
• Commonly made in the laboratory by solid-phase chemical synthesis,
these small bits of nucleic acids can be manufactured as single-
stranded molecules with any user-specified sequence, and so are vital
for artificial gene synthesis, polymerase chain reaction (PCR), DNA
sequencing, library construction and as molecular probes.
• In nature, oligonucleotides are usually found as small RNA molecules
that function in the regulation of gene expression (e.g. microRNA), or
are degradation intermediates derived from the breakdown of larger
nucleic acid molecules.
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 Principles of DNA biosensors Four chemical bases:
• Adenine(A), Guanine (G),
• Cytosine (C), and Thymine (T)
Nucleic acid hybridization
(Target Sequence)

ssDNA (Probe) (Hybridization) (Stable dsDNA)

 Bioreceptor

Display
 Biorecognition systems : Epigenetics
 It has been proposed that properly optimized integrated optical
resonators can be exploited for detecting epigenetic modifications
(e.g. DNA methylation, histone post-translational modifications) in
body fluids from patients affected by cancer or other diseases.
 Photonic biosensors with ultra-sensitivity are nowadays being
developed at a research level to easily detect cancerous cells within
the patient's urine.
 Different research projects aim to develop new portable devices that
uses cheap, environmentally friendly, disposable cartridges that
require only simple handling with no need of further processing,
washing, or manipulation by expert technicians.
 Biorecognition systems : Organelles

 Organelles form separate compartments inside cells and usually

perform function independently.
 Different kinds of organelles have various metabolic pathways and
contain enzymes to fulfill its function.
 Commonly used organelles include lysosome, chloroplast and
mitochondria.
 The spatial-temporal distribution pattern of calcium is closed related
to ubiquitous signaling pathway.
 Mitochondria actively participate in the metabolism of calcium ions
to control the function and also modulate the calcium related
signaling pathways.

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 Biorecognition systems : Cells

 Cells are often used in bioreceptors because they are sensitive to

surrounding environment and they can respond to all kinds of
stimulants.
 Cells tend to attach to the surface so they can be easily immobilized.
 Compared to organelles they remain active for longer period and the
reproducibility makes them reusable.
 They are commonly used to detect global parameter like stress
condition, toxicity and organic derivatives.
 Living Cell

Nourishment

Product
Bioreceptors

Bioreceptor

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 Bioreceptor

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 Biorecognition systems : Tissue
Tissues are used for biosensor for the abundance of enzymes existed.
Advantages of tissues as biosensors include the following:

 Easier to immobilize compared to cells and organelles

 The higher activity and stability
 The availability and low price
 The avoidance of tedious work of extraction, centrifuge and
purification of enzymes
 Necessary cofactors for enzyme to function exists
 There also exist some disadvantages of tissues, like the lack of
specificity due to the interference of other enzymes and longer
response time due to transport barrier.

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 Biorecognition systems : Lipids
Lipid: A lipid is chemically defined as a substance that is insoluble in
water and soluble in alcohol, ether, and chloroform.

Lipids are an important component of living cells. Together with

carbohydrates and proteins, lipids are the main constituents of plant and
animal cells.

Cholesterol and triglycerides are lipids. Lipids are easily stored in the
body. They serve as a source of fuel and are an important constituent of
the structure of cells.

Lipids include fatty acids, neutral fats, waxes and steroids. Compound
lipids comprise the lipoproteins, glycolipids and phospholipids.
 Biorecognition systems : Lipids

General structural
formula of a
glycerophospholipid. The
composition of the
specific molecule depends
on the chemical group
(designated R3 in the
diagram) linked to the
phosphate and glycerol
“head” and also on the
lengths of the fatty acid
“tails” (R1 and R2).
 Biorecognition systems : Lipids

Membrane lipids: The

membrane that surrounds a cell is
made up of proteins and lipids.
Depending on the membrane’s
location and role in the body,
lipids can make up anywhere
from 20 to 80 percent of the
membrane, with the remainder
being proteins. Cholesterol,
which is not found in plant cells,
is a type of lipid that helps stiffen
the membrane
 Biorecognition systems : Lipids
What are lipids soluble in?
Lipids are not soluble in water. They are non-polar and are thus soluble in
nonpolar environments like in choloroform but not soluble in polar
environments like water.
What do lipids consist of?
Lipids have mainly hydrocarbons in their composition and are highly
reduced forms of carbon. When metabolized, lipids are oxidized to release
large amounts of energy and thus are useful to living organisms.
Where do lipids come from?
Lipids are molecules that can be extracted from plants and animals using
nonpolar solvents such as ether, chloroform and acetone. Fats (and the
fatty acids from which they are made) belong to this group as do other
steroids, phospholipids forming cell membrane components etc.
 Biorecognition systems : Lipids
Phospholipid:
• Phospholipids are a class of lipids that
are a major component of all cell
membranes.
• They can form lipid bilayers because of
their amphiphilic characteristic.
• The structure of the phospholipid
molecule generally consists of two
Phospholipid Chemical makeup
hydrophobic fatty acid "tails" and a
with a of a single
hydrophilic "head" consisting of a hydrophilic head Phospholipid
phosphate group. and a
• The two components are joined together hydrophobic tail
by a glycerol molecule.
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 Biorecognition systems : Lipids
Liposome:
• A liposome is a spherical vesicle having at least one
lipid bilayer. The liposome can be used as a vehicle
for administration of nutrients and pharmaceutical
drugs. Liposomes can be prepared by disrupting
biological membranes.

• Liposomes are most often composed of

phospholipids, especially phosphatidylcholine, but
may also include other lipids, such as egg
phosphatidylethanolamine, so long as they are Scheme of a liposome
compatible with lipid bilayer structure. A liposome formed by
design may employ surface ligands for attaching to phospholipids in an
unhealthy tissue. aqueous solution.
 Biorecognition systems : Membrane/Cell surface receptors
• Cell surface receptors (membrane receptors,
transmembrane receptors) are receptors that are embedded
in the membranes of cells. They act in cell signaling by
receiving (binding to) extracellular molecules.
• They are specialized integral membrane proteins that allow
communication between the cell and the extracellular
space.
• The extracellular molecules may be hormones,
neurotransmitters, cytokines, growth factors, cell adhesion
molecules, or nutrients; they react with the receptor to
induce changes in the metabolism and activity of a cell. Membrane receptors
• In the process of signal transduction, ligand binding affects E = extracellular space
a cascading chemical change through the cell membrane. P = plasma membrane
I = intracellular space
Signal transduction mechanism in membrane lipids:

Signal transduction processes through membrane receptors involve the

external reactions, in which the ligand binds to a membrane receptor, and the
internal reactions, in which intracellular response is triggered.

Signal transduction mechanism in membrane lipids

Signal transduction through membrane receptors requires four parts:

1. Extracellular signaling molecule: an extracellular signaling molecule is

produced by one cell and is at least capable of traveling to neighboring cells.
2. Receptor protein: cells must have cell surface receptor proteins which
bind to the signaling molecule and communicate inward into the cell.
3. Intracellular signaling proteins: these pass the signal to the organelles
of the cell. Binding of the signal molecule to the receptor protein will
activate intracellular signaling proteins that initiate a signaling cascade.
4. Target proteins: the conformations or other properties of the target
proteins are altered when a signaling pathway is active and changes the
behavior of the cell.

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 Classification of Membrane receptors
Membrane receptors are mainly divided by structure and function into 3 classes: The ion
channel linked receptor; The enzyme-linked receptor; and The G protein-coupled receptor.

Ion channel linked receptors have ion channels for anions and cations, and constitute a large
family of multipass transmembrane proteins. They participate in rapid signaling events usually
found in electrically active cells such as neurons. They are also called ligand-gated ion
channels. Opening and closing of ion channels is controlled by neurotransmitters.

Enzyme-linked receptors are either enzymes themselves, or directly activate associated

enzymes. These are typically single-pass transmembrane receptors, with the enzymatic
component of the receptor kept intracellular. The majority of enzyme-linked receptors are, or
associate with, protein kinases.

G protein-coupled receptors are integral membrane proteins that possess seven

transmembrane helices. These receptors activate a G protein upon agonist binding, and the G-
protein mediates receptor effects on intracellular signaling pathways.
Biorecognition systems : Chemoreceptor

• A chemoreceptor (also known as chemosensor) is a specialized

sensory receptor cell which transduces (responds to) a chemical
substance (endogenous or induced) and generates a biological
signal.

• This signal may be in the form of an action potential if the

chemoreceptor is a neuron (nerve cell), or in the form of a
neurotransmitter that can activate a nearby nerve fiber if the
chemosensor is a specialized sensory receptor cell, such as the
taste receptor in a taste bud or in an internal peripheral
chemoreceptor such as the carotid body (ex, in chemotherapy).
Biorecognition systems : Chemoreceptor
• In more general terms, a chemosensor
detects toxic or hazardous chemicals in
the internal or external environment of
the human body (e.x. chemotherapy) and
transmits that information to the central
nervous system, (and rarely the
peripheral nervous system), in order to
expel the biologically active toxins from
the blood, and prevent further
consumption of alcohol and/or other
acutely toxic recreational intoxicants.
What are Neurotransmitters and Neuroactive Peptides
• Communication of information between neurons is accomplished by
movement of chemicals across a small gap called the “synapse”.
• Chemicals, called neurotransmitters, are released from one neuron at
the pre-synaptic nerve terminal.
• Neurotransmitters then cross the synapse where they may be accepted by
the next neuron at a specialized site called a “Receptor“.
• The action that follows activation of a receptor site may be either
depolarization (an excitatory post-synaptic potential) or
hyperpolarization (an inhibitory post-synaptic potential).
• A depolarization makes it MORE likely that an action potential will fire,
a hyperpolarization makes it LESS likely that an action potential will
fire.
Neurons network
Neurotransmitters
Function of Receptors
• Plants have various mechanisms to perceive danger in their environment. Plants are
able to detect pathogens and microbes through surface level receptor kinases (PRK).

• Taste receptors in the gustatory system: The primary use of gustation as a type of
chemoreception is for the detection of tasteants. Aqueous chemical compounds come
into contact with chemoreceptors in the mouth, such as taste buds on the tongue, and
trigger responses.

• Particular chemoreceptors, called ASICs, detect the levels of carbon dioxide in the
blood. To do this, they monitor the concentration of hydrogen ions in the blood,
which decrease the pH of the blood.

• Peripheral chemoreceptors: consists of aortic and carotid bodies. Aortic body

detects changes in blood oxygen and carbon dioxide, but not pH, while carotid body
detects all three.
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Receptor Binding
• Neurotransmitters will bind only to specific receptors on the post-synaptic membrane
that recognize them.

Inactivation of Neurotransmitters:
The action of neurotransmitters can be stopped by four different mechanisms:
1. Diffusion: the neurotransmitter drifts away, out of the synaptic cleft where it can no
longer act on a receptor.
2. Enzymatic degradation (deactivation): a specific enzyme changes the structure of
the neurotransmitter so it is not recognized by the receptor. For example, acetyl-
cholinesterase is the enzyme that breaks acetylcholine into choline and acetate.
3. Glial cells: astrocytes remove neurotransmitters from the synaptic cleft.
4. Re-uptake: the whole neurotransmitter molecule is taken back into the axon terminal
that released it. This is a common way the action of norepinephrine, dopamine and
serotonin is stopped…these neurotransmitters are removed from the synaptic cleft so
they cannot bind to receptors.
Biorecognition systems : Immunoreceptor

• Cells of the immune system communicate with their environment

through immunoreceptors. These receptors often harbor
intracellular tyrosine residues, which, when phosphorylated upon
receptor activation.
• Immune receptor signaling, critical for proper immune response,
involves signaling pathways mediated by specific tyrosine
residues that are phosphorylated upon receptor activation.
• Neutrophils (also called polymorphonuclear leukocytes: PMNs)
are the most abundant type of white blood cell in the human body.
Their primary task is to protect the body from harmful microbial
infections, particularly those exerted by bacterial and fungal
pathogens. These defensive mechanism needs immunoreceptors.
Classification of Immunoreceptor

• Several classes of cell surface receptors on neutrophils are

involved in cellular activation and intracellular signal
transduction.

• These include G protein-coupled receptors (GPCRs), cytokine and

chemokine receptors, adhesion receptors (e.g. integrins or
selectins) and pattern recognition receptors (PRRs) such as Toll-
like receptors (TLRs) or C-type lectin receptors (CLRs).

• Additionally, modulation of immune responses by neutrophils is

regulated through activating and inhibitory immunoreceptors.

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Immunoreceptor function in disease

• Neutrophils are dynamic and versatile cells with a primary role in

the host defense against bacteria, but have also been shown to be
important in other inflammatory diseases, including
autoimmunity, and accumulating evidence suggest that they also
play a role in cancer.
• As above described, neutrophils are equipped with an impressive
variety of immunoreceptors that have the capacities to control and
fine-tune the many effector functions of these cells through
largely defined canonical activating and inhibitory signaling
pathways.
Immunoreceptor function in disease
Immunoreceptors play a key role the following disease conditions such as

1. Infection: There is convincing evidence that at least some of the activating

immunoreceptors on neutrophils contribute to the recognition of pathogens, thereby
promoting activation of some or all of the various neutrophil effector functions, and which
ultimately lead to the elimination of the infection.
2. Inflammation: The innate immune system is crucial for the first line of defense against
pathogens. Binding of PAMPs to PRRs triggers inflammatory responses that are needed to
combat pathogens.
3. Autoimmunity: Autoimmune diseases are characterized by detrimental and harmful
immune activation directed against auto-antigens and immunity towards host tissues.
Abnormal signaling through activating or inhibitory receptors may certainly contribute to
autoimmune development and maintenance.
4. Cancer: Apart from macrophages, also neutrophils may positively or negatively
affect tumor progression.
Immobilization of Biomolecules

Immobilization
 Molecules may be immobilized either passively through;
o Hydrophobic
o Ionic interactions
o Covalently by attachment to activated surface groups.
 Non-covalent surfaces are effective for many applications;
however, passive adsorption fails in many cases.
 Covalent immobilization is often necessary for binding of
molecules that do not adsorb, adsorb very weakly, or adsorb
with improper orientation and conformation to non-covalent
surfaces.
 Covalent immobilization may result in better biomolecule
activity, reduced nonspecific adsorption, and greater stability.
 Immobilization
Immobilization reaction should have several characteristics;

o Firstly, the reaction should occur rapidly and therefore allow the use of
low concentrations of reagents for immobilization.
o The chemistry should require little, if any, postsynthetic modification
of ligands before immobilization to maximize the number of
compounds that can be generated by solution or solid-phase synthesis
and minimize the cost of these reagents.
o Immobilized ligands must be in an oriented and homogeneous manner.
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 Immobilization
The immobilization process should occur selectively in the presence of
common functional groups, including amines, thiols, carboxylic acids,
and alcohols.

 Amino-NH2,
 Carboxy-COOH,
 Aldehyde-CHO,
 Thiol-SH,
 Hydroxyl-OH
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THANK YOU

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