• A biological recognition element or bioreceptor is generally

consists of an immobilized biocomponent that is able to detect the

specific target analyte.

)(Pr)()( PoductSSubstrate EEnzyme

1- Enzymes

Proteins composed by a number of amino acid residues

The only difference; possess catalytic activity

Biological catalysis that facilitate the conversion of substrate

into products by lowering the activation energy of the reaction

Biomolecules

Used for detection of phenols, pesticides, some food additives etc.

depending on their inhibition mechanisms

Biological signal mechanisms

Enzyme based sensor contains enzyme as bioreceptor and this

enzyme is specific to target analyte from sample matrix.

High specific enzyme–substrate interactions and high turnover

rates of biocatalysts causes sensitive and specific detection.

Currently, biosensor based on enzyme inhibition is trend

application. Especially in clinical field, inhibitors can be considered

as toxic compound besides the drug.

Biological signal mechanisms

• Enzymes are first biomolecular recognition element used in

biosensors and enzyme based biosensor is first introduced by

Clark and Lyons in 1962 for amperometric enzyme electrode

for sensing the glucose.

Glucose oxidase is first used in

enzyme-based biosensor for

sensing glucose.

Table . Enzymes as biomolecules in development of various types of biosensors

Enzyme

Advantages

It is possible to reuse the enzyme

Allows easy separation from reaction media

pH, temperature, storage stability and catalytic properties can

be enhanced

Practical limitations

The yield of protein binding is rarely quantitative

The cost of the support may exceed the cost of the enzyme

Decrease in enzyme activity due to steric hindrance and mass

transfer limitations.

The proportion of active enzyme rarely exceeds 5-10 % w/w

2- Whole-cell based sensor

• Whole-cell based sensor depends on complex cellular

functions as well as enzyme catalytic reactions.

• Proteins present in cells can also be used as bioreceptors for

the detection of specific analyte.

3- Antibody based sensor (immunosensor)

• Depends on antibody-antigen biorecognition.

• An antibody is generally composed of 2 heavy chains and 2

light chains. Each chain has constant and variable parts.

Variable fragment (Fv) is specific to the antigen which binds

to its antigen.

Fv

4- Nucleic acid based sensor

Use nucleic acids to form double stranded DNA (dsDNA) by

the highly specific affinity binding reaction between two

single strand DNA (ssDNA) chains. This type of biosensor

uses an immobilized ssDNA to complementary to target.

5- Aptamers

• Besides the nucleic acid does base pairing, aptamer is self-annealing and create specific three dimensional structure.

• Are synthetic strands of nucleic acid which can be designed to recognize amino acids, oligosaccharides, peptides and proteins.

• Aptamers were reported for the first time in the early 1990s where described as artificial nucleic acid ligands.

5- Aptamers

• Aptamer properties such as their high specificity, small size,

modification, regenerability and immobilization versatility

• Conformational change induced by the target binding have been

successfully exploited to optimize a variety of bio-sensing

formats.

• An aptamer has few advantages over antibody based biosensor

such as high binding efficiency, avoiding the use of animal (i.e

reduced ethical problem), smaller and less complex, and etc.

• Also, chemical modification provides immobilization of aptamers

to various solid supports. Aptamers can be easily chemically

modified by various chemical tags including fluorescence probes,

quenchers, electrochemical indicators, nanoparticles or enzymes.

This modification allows to immobilize aptamers to various solid

supports

Table. Examples of targets of aptamers

6- Polymer based Biosensor

• Molecular Imprinting Polymer (MIP) based sensor MIPs

typically contain template molecules, functional monomers,

cross-linking reagents.

Figure . Imprinting Polymerisation

6- Polymer based Biosensor

Figure . Imprinting Polymerization

To give artificial recognition functions, complementary microcavities are

created for specific targets, like the “key-and-lock”.

As biomimetic synthetic receptors, MIPs have specific cavities for targets,

superior to natural antibody recognition.

Cross-linked polymer formed around a molecule that acts as a template,

template subsequently removed. Imprints containing functional groups

complementary to those of template remain in the polymer

Figure 3. Illustration of the fabrication and application of MIPs-based electrochemical

biosensors (Gui et al., 2018)

When templates are produced from polymers at the molecular level, MIPs can

recognize and rebind targets with high specificity and affinity, much superior to

natural receptors. (electrochemical sensor covering with MIP has;

The highly mechanical/thermal stability, excellent specificity and sensitivity of

MIPs-based ECBSs for targets indicate a greater prospect for high-quality sensing

applications, over traditional instrument techniques and other types of sensors.

Why we need immobilization?

Continuous

processing

Reuse of

biomolecules

Low

residence

time

Stabilization

Immobilization Methods

Involve covalent bond formation Do not involve covalent bond formation

Native composition of biomolecule remains unaltered More stable immobilized biomolecule

Immobilization

Methods

Physical Methods

Entrapment

Adsorption Encapsulation

Chemical Methods

Covalent cross-linking

Covalent binding

The carrier-free immobilization

methods such as cross-linked

enzyme aggregates (CLEA)

Groups in the structure of the enzyme

• Alpha carboxyl group at “C’’ terminal of enzyme

• Phenol ring of Tyrosinase

• Thiol group of Cysteine

• Alpha amino group at “N’’terminal of enzyme

• Hydroxyl group of Serine and Threonine

Methods of covalent bonding

1. Diazzoation

2. Peptide Bond

3. Poly Functional Reagents

1. Diazoation

Covalent bonding is consisted between amino group of

support and tyrosil or histidyl group of enzyme.

2. Peptide Bond

Covalent bonding is consisted between amino or carboxyl

groups of support and amino or carboxyl groups of enzyme.

3. Poly Functional Reagents

Covalent bonding is consisted between amino group of

support and amino group of the enzyme.

Bi-functional or multifunctional reagent is used.

Advantage of Covalent Bonding Method

Preparation is easy

Strong bonds

Ideal mass production

Useful for enzyme

Stable enzyme-support complex

Disadvantage of Covalent Bonding Method

High cost

Funciton of enzyme loss.

Crosslinking

Another name is copolymerization.

Covalent bond is used.

Between various group of enzyme

via poly functional reagent.

No support material

• Glutaraldehyde, glyoxal, hexamethylenediamine and

diazonium is the most common used

Advantage of Crosslinking Method

Preparation is simple.

Strong chemical bonds.

Used for physically adsorbed enzyme or proteins.

Cheap

Disadvantage of Crosslinking Method

Control is diffucult

A large amount of enzyme

Low enzyme activity

Do not use for pure enzyme

Entrapment

Figure : Immobilization of enzyme using the entrapment technique

• Entrapment means physical enclosure of biomolecule in a small space

• A polymeric gel is prepared in a solution containing the biomaterial.

• The biomaterial is thus entrapped within the gel matrix

• Matrices commonly used are chemical polymers such as calcium

alginate, carrageenan, polyacrylamide, and sol-gel.

ADVANTAGES DISADVANTAGES

• Simplest method for

biomolecule immobilization

• Gentle method that

requires no chemical

modification and only mild

reaction condition is

applied thus minimizing

loss of bio-activity

• Large barriers causes

inhibition of substrate

diffusion

• Slows reaction

• Decreases response time

• lowers sensitivity and

detection limit

• Leakage of enzymes

through pores

• loss of bioactivity through

the pores in the gel.

Microencapsullation

Figure : Immobilization of enzymes using the encapsulation technique

• Involves the formation of spherical particle called as

“microcapsule” in which a liquid or suspension of biocatalyst is

enclosed within a semi permeable polymeric membrane.

• It provides the close contact between biomaterial and

transducer.

ADVANTAGES DISADVANTAGES

• Close attachment between

biomaterial and transducer

• It is very adaptable

• It is very reliable

• Attachment is weak thus

leaching of enzyme from

support

• Encapsulated biomaterial

is very susceptible to

environmental changes

(pH, temperature, ionic

strength)

• Suitable for short-term

investigations

• Limited lifetime

Adsorption

• Figure : Immobilization of enzymes using the adsorption technique.

• Oldest and simplest method of biomolecule immobilization

• The method depends on non-specific physical interaction

between the biomolecules and the surface of the matrix.

• Many substances adsorb biomolecules on their surfaces.

Hydrophobic forces

• Hydrophobic

groups intended to

interact with

hydrophobic surface

to minimize the

surface energy

• Strong force

Electrostatic forces

• Attraction between

opposite charges

• Strong force

Van der waals forces

• Fluctuations in electron clouds

around molecules oppositely

polarized neighboring atoms

• Weak force

Hydrogen bonds

• Hydrogen shared

between electronegative

atoms (N, O)

• Stronger than Van der

waals force

Driving forces for physical adsorption

Figure: Schematic diagram showing the effect of soluble enzyme

concentration on the activity of enzyme immobilised by adsorption

to a suitable matrix.

Ionic

Strengh

pH

Subsrate

concentration

Temperature

ADVANTAGES DISADVANTAGES

• No pore diffusion limitation,

• Easy to carry out,

• No reagents required /

minimal preparation

• No clean-up step

• Less disruption to the

enzymes

• Low cost

• Non-specific

• Attachment is weak

• Adsorbed biomaterial very

susceptible to

environmental changes (pH,

temperature, ionic strength)

• Suitable for short-term

investigations

• Limited lifetime

Why most of the commercial medical biosensors use

physical adsorption method for immobilization of

biomolecules?

Minimum modification to biomolecules

(preserve function)

Simple and easy to perform

Low cost Fast

Immobilization

• Immobilization of biomolecules with

eachother

•Immobilization of biomolecules on a working

electrode

•Immobilization of biomolecules to an

appropriate support material: solid insoluble

matrix where biomolecules can be attached to

Ideal Support Material

Inert

Large surface are

Physical Strength

Optimum quality

Provides the

optimal micro-environment

Having functional groups

Enhance biomolecule

specificity

Carriers used for immobilization by adsorption

• Alginate

• Chitosan

• Chitin

• Collagen

Natural Polymers:

• Polyvinyl chloride (PVC)

• Diethylaminoethyl cellulose (DEAE-C)

Synthetic Polymers:

• Zeolites

• Ceramics

• Glass

• Slica

• Activated carbon

Inorganic Materials: