biosensor and bochips - Principles, Fabrication and Applications

Biosensors and Biochips:

Principles, Fabrication and Applications




Biosensors

A biosensor is defined by IUPAC (1997) as:
‘‘A device that uses specific biochemical reactions mediated by
isolated enzymes, immunosystems, tissues, organelles or whole
cells to detect chemical compounds usually by electrical, thermal
or optical signals’’.

Almost all biosensors are based on a three-component system: a
support, a biological recognition element (ligand) that facilitates
specific binding or biochemical reaction of a target, and a signal
conversion unit (transducer).

Biosensors

Miners used birds to reveal the presence of toxic gases in mines.
Birds like canaries die if exposed to low gas concentrations. This is
one early example of a
disposable ON/OFF biosensor. The gas is
the analyte, the canary acts as the biological active species, and the

detection limit is the minimal gas concentration that can kill the bird.



Gas concentration
is below
the detection limit
The bird lives
Gas concentration
is above
the detection limit
The bird dies
Biosensors



An even earlier example of
disposable ON/OFF biosensor
is the
food taster, usually a
slave that tested the king food
for poisons (like Remy in
Ratatouille).
Of course, the food taster was
disposed if deadly poison was
present in the food.

Biosensors

A more recent example of
disposable ON/OFF biosensor
is the
mine detector, a rat
trained to find mines. Its light
weight allows it for walking
on mines without any risk of
explosion.
Of course, the mine tester is
disposed if the mine is too
sensitive to pressure.

Biosensors – History

1956 Clark published a paper on the oxygen electrode, and used the
term “enzyme electrode”. The concept of biosensor was born.

1969 Guilbault and Montalvo described the first potentiometric
enzyme electrode, based on the use of urease to detect urea.

1975 The Yellow Springs Instrument Co. launched a glucose
biosensor
based on the amperometric detection of hydrogen
peroxide.

1976 The BIOSTATOR (electrochemical glucose biosensor in
artificial pancreas) was invented.

1982 The first needle-type enzyme electrode for subcutaneous

implantation of glucose biosensors was reported.

1987 MediSense launched a pen-sized meter for home blood-
glucose monitoring.

Biosensor requirements

To satisfy a large number of applications, a biosensor
should be:

easy to manufacture in large numbers

easy to use


cheap if disposable

reusable if expensive

fast in response

able to perform multiple tests at the same time
(microarray)

Biosensor Structure

Almost all biosensors are based on a three-component system: a
support, a biological recognition element (ligand) that facilitates
specific binding or biochemical reaction of a target, and a signal
conversion unit (

transducer).



Biosensors – Transducer

The transducer converts the biochemical interactions into a
measurable electronic signal.

Electrochemical, electrooptical, acoustical, and
mechanical transducers
are among the many types found
in biosensors.

The transducer works either
directly or indirectly.

Direct Transducers

Direct detection sensors, in which the biological interaction is
directly measured in real time, typically use non-catalytic ligands
such as cell receptors or antibodies.

The most common direct detection
biosensor systems employ surface
plasmon resonance
.


Quartz resonator transducers that measure

changes in acoustic resonance and opto–
mechanical biosensors (
microcantilevers) are
other direct recognition sensors.

Diode
array
Laser
Prism
θ
Metal
Polymer
Flow cell
Indirect Transducers

The second class of transducers,
indirect detection sensors, relies
on secondary elements that are often catalytic elements such as
enzymes. Some examples of secondary elements are:

Electrochemical transducers to measure the oxidation or reduction
of an electroactive compound on the secondary ligand.

Fluorescence, measuring fluorescence of the secondary ligand.

Fluorescence Resonance Energy Transfer (FRET), a process
where energy from an excited fluorophore is transferred to a
neighboring acceptor molecule.

Biosensors – Ligands

The most important characteristics for ligands are affinity and
specificity. Ligands can be:

Molecules, macromolecules, whole cells, tissues, microorganisms.

Antibodies are common recognition elements because of high
specificity, versatility, and strong binding to the antigen.

Aptamers, protein-binding DNA or RNA (K
d
10
8
–10
9
M) amenable
for high-throughput screening, are easy to manufacture. However,
they can only be used for targets that bind nucleic acids.

Peptides, combined binding agents derived from low-affinity
ligands and combinatorial chemistry ligands for biosensors.


Substrates





Silicon wafers

Gold foil Mica Glass slides





Graphite sheets Nitrocellulose Nylon membrane


Biomolecule Immobilization

Biomolecules can be immobilized on the support in two main ways:

PASSIVE ADSORPTION
Adsorption is thermodynamically spontaneous at low protein
concentration. Adsorption is almost irreversible on hydrophobic
surfaces; desorption is possible on hydrophilic surfaces.

COVALENT IMMOBILIZATION
The surface must be functionalized; the functional groups can be
separated from the surface using a spacer;
the functional groups
can be selective or non-selective

Both methods can be performed either directly on the support surface
or on an additional layer deposited on the support.

Proteins on Surfaces

Cationic surface Anionic surface Hydrophobic surface
Polar residue
Non–Polar residue
Negatively charged residue
Positively charged residue
Hydrophilic surface
Surface Modification



Biomolecule Immobilization



Substrate surface
Activated
Surface
Biofunctionalized
Surface
Functionalized
Surface
Electrostatic Interaction Covalent attachment
Avidin/Streptavidin
Bioactive compound
Charged species
Reactive functional groups
Biotin

Affinity Interaction
Effect on Bioactivity



Increased immobilization
because of branched spacer
Reduced immobilization
due to overfunctionalization
Reduced bioactivity
because of overcrowding
Reduced nonspecific
adsorption
Increased mobility
due to spacer flexibility
Surface induced
denaturation
P P
Covalent Binding to Substrates




HS
hν
O
O
N
N
CF

3
hν
CF
3
NN
O
O
S
Covalent Binding to Substrates







O
N
O
O
O
H
2
N
N
H
O
O
H
2

N
N
NaCNBH
3
N
H
Covalent Binding to Substrates





H
NN
O
O
HS
O
H
NN
O
O
O
S
S
HS
O
O
S
O

O
S
S
S
HS
S
S
N
Covalent Binding to Substrates










N
O
O
O
N
O
O
O
N
3
N

N
N
N
3
N
N
N
Covalent Binding to Substrates



biotinylated
antibody
biotin
O
HN NH
S
H H
OH
O
Biosensor Fabrication

Many high-level technologies have been developed and consolidated
for microelectronic industry, and can be used for the production of
micro- and nano-scaled biosensors.

Screen printing

Liquid-handling techniques (syringe-type processes, ink-jet
printing, etc.)

Photolithography

Nanopatterning

Screen Printing or Serigraphy

A screen is made of a piece of porous, finely woven fabric (originally
silk). A negative image is made by masking areas of the screen; ink
is transferred to the substrate by capillary action.



Syringe–type Processes