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DEFINITION

• A biosensor may be defined as a device
  incorporating a biologically active component in
  intimate contact with a physico-chemical
  transducer and an electronic signal processor.

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Analyte of interest
Interfering species
Biocomponent
Transducer
Signal
Processor
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So what is an biosensor?
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HISTORY

• 1962, Clark and Lyons
• 1967, Updike and Hicks
• 1969, Guilbault
• 1972, Reitnauer
• 1975, Yellow Springs Instrument
• 1975, Janata
• 1979, Danielsson

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BIOCOMPONENTS

• Enzymes
• Antibodies
• Membranes
• Organelles
• Cells
• Tissues
• Cofactors

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TRANSDUCERS

• Electrochemical
• Optical
• Piezo-electric
• Calorimetric
• Acoustic

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BIOSENSOR TYPES

• Enzyme/metabolic biosensors
• Enzyme and cell electrodes
• Bioaffinity sensors
• Antibodies
• Nucleic acids
• Lectin

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Enzyme/Metabolic Sensors

• Enzymes are biological catalysts. There are five
  main classes of enzymes.
• Oxidoreductases
• Transferases
• Hydrolases
• Lyases
• Isomerases

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Oxidoreductases

• Dehydrogenases
• Oxidases
• Peroxidases
• Oxygenases

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Enzyme/Metabolic Sensors

• Substrate Enzyme

Substrate-enzyme complex
Product Enzyme
Substrate consumption/product liberation is
measured and converted into quantifiable signal.
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Bioaffinity Sensors

• These sensors are based on binding interactions
  between the immobilised biomolecule and the
  analyte of interest.
• These interactions are highly selective.
• Examples include antibody-antigen interactions,
  nucleic acid for complementary sequences and
  lectin for sugar.

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Analyte of interest (antigen)
Antibody
Interfering species
Antibody-antigen complex
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Transducers

• Electrochemical
• Potentiometric
• Amperometric
• Conductimetric

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POTENTIOMETRIC BIOSENSORS

• In potentiometric sensors, the zero-current
  potential (relative to a reference) developed at
  a selective membrane or electrode surface in
  contact with a sample solution is related to
  analyte concentration.
• The main use of potentiometric transducers in
  biosensors is as a pH electrode.

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POTENTIOMETRIC BIOSENSORS

• E Eo RT/nF lnanalyte
• Eo is a constant for the system
• R is the universal gas constant
• T is the absolute temperature
• z is the charge number
• F is the Faraday number
• lnanalyte is the natural logarithm of the
  analyte activity.

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POTENTIOMETRIC BIOSENSORS

• The best known potentiometric sensor is the Ion
  Selective Electrode (ISE).
• Solvent polymeric membrane electrodes are
  commercially available and routinely used for the
  selective detection of several ions such as K,
  Na, Ca2, NH4, H, CO32-) in complex biological
  matrices.
• The antibiotics nonactin and valinomycin serve as
  neutral carriers for the determination of NH4
  and K, respectively.

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Ag/AgCl reference electrode
Internal aqueous filling solution
Liquid ion exchanger
Membrane/salt bridge
Porous membrane containing ionophore
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POTENTIOMETRIC BIOSENSORS

• ISEs used in conjunction with immobilised enzymes
  can serve as the basis of electrodes that are
  selective for specific enzyme substrates.
• The two main ones are for urea and creatinine.
• These potentiometric enzyme electrodes are
  produced by entrapment the enzymes urease and
  creatinase, on the surface of a cation sensitive
  (NH4) ISE.

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POTENTIOMETRIC BIOSENSORS
urease
Urea H2O H
2NH4 HCO3-
creatininase
Creatinine H2O
N-methylhydantoin NH4
penicillinase
Penicillin
Penicillonic Acid
In contact with pH electrode.
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AMPEROMETRIC BIOSENSORS

• With amperometric sensors, the electrode
  potential is maintained at a constant level
  sufficient for oxidation or reduction of the
  species of interest (or a substance
  electrochemically coupled to it).
• The current that flows is proportional to the
  analyte concentration.
• Id nFADsC/d

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Auxiliary Electrode
(e.g. Pt wire)
Working Electrode
Reference Electrode
(e.g. Pt, Au, C)
(e.g. Ag/AgCl, SCE)
Buffer solution (e.g. Tris, DPBS,
Citrate) incorporating electrolyte (e.g. KCl,
NaCl)
e flow
Stirbar
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Example Glucose O2
Glucose
Gluconic Acid H2O2
Oxidase
The product, H2O2, is oxidised at 650mV vs a
Ag/AgCl reference electrode. Thus, a potential
of 650mV is applied and the oxidation of H2O2
measured. This current is directly proportional
to the concentration of glucose.
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I (nA)
150
100
50
0
5
10
15
20
Glucose, mM
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AMPEROMETRIC BIOSENSORS

• Amperometric enzyme electrodes based on oxidases
  in combination with hydrogen peroxide indicating
  electrodes have become most common among
  biosensors.
• With these reactions, the consumption of oxygen
  or the production of hydrogen peroxide may be
  monitored.
• The first biosensor developed was based on the
  use of an oxygen electrode.

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Clark Oxygen Electrode
-

Electrode body
Silver anode
KCl soln.
Polyethylene membrane
Platinum cathode
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AMPEROMETRIC BIOSENSORS

• The drawback of oxygen sensors is that they are
  very prone to interferences from exogenous
  oxygen.
• H2O2 is more commonly monitored. It is oxidised
  at 650mV vs. a Ag/AgCl reference electrode.
• At the applied potential of anodic H2O2
  oxidation, however, various organic compounds
  (e.g. ascorbic acid, uric acid, glutathione,
  acetaminophen ...) are co-oxidised.

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AMPEROMETRIC BIOSENSORS

• Various approaches have been taken to increase
  the selectivity of the detecting electrode by
  chemically modifying it by the use of
• membranes
• mediators
• metallised electrodes
• polymers

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AMPEROMETRIC BIOSENSORS
1. Membranes. Various permselective membranes
have been developed which controlled species
reaching the electrode on the basis of charge
and size.
Examples include cellulose acetate (charge and
size), Nafion (charge) and polycarbonate (size).
The disadvantage of using membranes is, however,
their effect on diffusion.
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AMPEROMETRIC BIOSENSORS
2. Mediators Many oxidase enzymes can utilise
artificial electron acceptor molecules, called
mediators.
A mediator is a low molecular weight redox
couple which can transfer electrons from the
active site of the enzyme to the surface of the
electrode, thereby establishing electrical
contact between the two.
These mediators have a wide range of structures
and hence properties, including a range of redox
potentials.
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AMPEROMETRIC BIOSENSORS
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AMPEROMETRIC BIOSENSORS

• Examples of mediators commonly used are
• Ferrocene (insoluble)
• Ferrocene dicarboxylic acid (soluble)
• Dichloro-indophenol (DCIP)
• Tetramethylphenylenediamine (TMPD)
• Ferricyanide
• Ruthenium chloride
• Methylene Blue (MB)

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AMPEROMETRIC BIOSENSORS
3. Metallised electrodes The purpose of using
metallised electrodes is to create conditions in
which the oxidation of enzymatically generated
H2O2 can be achieved at a lower
applied potential, by creating a highly catalytic
surface.
In addition to reducing the effect of
interferents, due to the lower applied potential,
the signal-to-noise ratio is increased due to an
increased electrochemically active area.
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AMPEROMETRIC BIOSENSORS
Metallisation is achieved by electrodepositing
the relevant noble metal onto a glassy carbon
electrode using cyclic voltammetry.
Successful results have been obtained from a few
noble metals - platinum, palladium, rhodium and
ruthenium being the most promising.
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Glassy carbon electrode
Metallised GCE
Glassy carbon electrodes do not catalyse the
oxidation of hydrogen peroxide.
GCEs metallised with ruthenium, rhodium,
palladium or platinum do.
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AMPEROMETRIC BIOSENSORS
4. Polymers As with membranes, polymers are used
to prevent interfering species from reaching the
electrode surface. Polymers differentiate on the
basis of size and charge.
An example is that of polypyrrole. A polypyrrole
film has to be in the reduced state to become
permeable for anions. If the film is oxidised,
no anion can permeate.
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AMPEROMETRIC BIOSENSORS

• Examples of commonly used polymers are
• polypyrrole
• polythiophene
• polyaniline
• diaminobenzene
• polyphenol

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Electrochemical Transducers
3. Conductimetric Conductimetric methods use
non-Faradaic currents. In conductimetric
transducers the two electrodes (working and
reference) are separated from the measuring
solution by a gas-permeable membrane. The
measured signal reflects the migration of all
ions in the solution. It is therefore
non-specific and may only be used for samples of
identical conductivity.
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K
K
K
A-
A-
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OPTICAL BIOSENSORS

• The area of biosensors using optical detection
  has developed greatly over the last number of
  years due mainly to the inherent advantages of
  optical systems.
• The basis of these systems is that enzymatic
  reactions alter the optical properties of some
  substances allowing them to emit light upon
  illumination.
• Means of optical detection include fluorescence,
  phosphorescence, chemi/bioluminescence...

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OPTICAL BIOSENSORS

• Advantages of optical biosensors include
• due to fibre optics, miniaturisation is possible
• in situ measurements are possible
• in vivo measurements are possible
• diode arrays allow for multi-analyte detection
• signal is not prone to electromagnetic
  interference

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OPTICAL BIOSENSORS

• Disadvantages include
• ambient light is a strong interferent
• fibres are very expensive
• indicator phases may be washed out with time

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OPTICAL BIOSENSORS

• Fibre optics are a subclass of optical waveguides
  which operate using the principle of total
  internal reflection.
• Light incident on the interface between two
  dielectric media will be either reflected or
  refracted according to Snells Law.

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OPTICAL BIOSENSORS
A
Cladding
Core
B
B
Total Internal Reflection
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OPTICAL BIOSENSORS

• If light is entered into a fibre (surrounded by a
  medium of lower refractive index) at a shallow
  enough angle, the light will be confined within.
• Thus, the optical fibres consist of a core of
  high refractive index surrounded by a cladding of
  slightly lower refractive index, with the whole
  fibre protected by a non-optical jacket.

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OPTICAL BIOSENSORS
Jacket
qmax
Cladding
Core
Cladding
Jacket
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OPTICAL BIOSENSORS

• Light input, and hence output, is dependent on
  the diameter of the fibre.
• As a very small diameter is required for flexible
  fibres, this size is a limiting factor in the
  fabrication of the fibres.
• For this reason, fibres are made from bundles
  which have the advantage of efficient light
  collection and flexibility.
• Fibre bundles of 8, 16 and more fibre strands are
  available.

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OPTICAL BIOSENSORS

• Generally, fibre-optic based biosensors employ
  fluorescence or chemiluminescence as the light
  medium.
• This is due to the fact that fluorescence is
  intrinsically more sensitive than absorbance.
• It is also more flexible due to the fact that a
  great variety of analytes and influences are
  known to change the emission of particular
  fluorophores.

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OPTICAL BIOSENSORS

• There are basically two different configurations
  used at the tip of the fibre-optic probe
• the distal cuvette configuration
• waveguide binding configuration

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OPTICAL BIOSENSORS

• The distal cuvette configuration involves
  immobilisation of detection molecules in a
  porous, transparent medium at the fibre tip.
• The fluorescence changes when the analyte
  diffuses and is bound.
• Excitation comes from out of the fibre and
  emission is coupled back into the fibre.

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OPTICAL BIOSENSORS
Fibre-optic probe
Distal cuvette
Detection molecule
Analyte
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OPTICAL BIOSENSORS

• The waveguide binding tip configuration involves
  the binding of fluorescent-labelled detector
  molecules (e.g. antibodies) to covalently
  attached analyte molecules on the fibre surface.
• As the label is close to the surface it is
  excited by the evanescent wave emanating from the
  fibre and the resulting fluorescence is coupled
  back into the fibre.
• Free analyte competes for the binding sites on
  the recognition molecules, permitting them to
  diffuse away from the surface with a resultant
  decrease in fluorescence.

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OPTICAL BIOSENSORS
Fluorescent-labelled recognition molecule
Free antigen
Immobilised analyte
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Piezoelectric Transducers

• The principle of this sensor type is based on the
  discovery of there being a linear relationship
  between the change in the oscillating frequency
  of a piezoelectric (PZ) crystal and the mass
  variation on its surface.
• Sauerbrey discovered in 1959 that the change in
  mass is inversely proportional to the change in
  frequency of the resonating crystal (usually at
  MHz frequencies).

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Piezoelectric Transducers

• DF -2.3x106F2DM/A (Sauerbrey equation)
• The change in mass occurs when the analyte
  interacts specifically with a biospecific agent
  immobilised on the crystal surface.
• The crystal may be coated with antibodies,
  enzymes or organic materials.
• Frequency changes smaller than 1MHz may be
  measured providing nanogram sensitivity.

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Quartz wafer
Gold
Electrical contacts
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SAW Transducers

• Surface acoustic wave (SAW) devices operate by
  the propogation of acoustoelectric waves, either
  along the surface of the crystal or through a
  combination of bulk and surface.
• The oscillation of the crystal in SAW devices is
  greater, by at least a factor of ten, than the
  oscillation of the crystal used in PZ devices.

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Calorimetric Transducers

• Enzyme-catalysed reactions exhibit the same
  enthalpy changes as spontaneous chemical
  reactions.
• Considerable heat evolution is noted
  (5-100kJ/mol).
• Thus, calorimetric transducers are universally
  applicable in enzyme sensors.

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Calorimetric Transducers

• The thermal biosensors constructed have been
  based on
• direct attachment of the immobilised enzyme or
  cell to a thermistor
• Immobilisation of the enzyme in a column in which
  the thermistor has been embedded.
• DT nDH/cp

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Enzyme Substrate -DH (kJ/mol) Catalase Hydrog
en peroxide 100.4 Cholesterol oxidase Cholesterol
52.9 Glucose oxidase Glucose 80.0 Hexokinase
Glucose 27.6 Lactic dehydrogenase Pyruvate 62
.1 b - Lactamase Penicillin G 67.0 Urease Ure
a 6.6 Uricase Uric acid 49.1
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Recorder
Bridge/Amplifier
Sample
Thermistor
Buffer stream
Enzyme reactor
Heat exchanger
polyurethane insulation
Aluminium block