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Subtopic
Chapter 6 – Introduction to biosensors
– Type of transducers
BIOSENSORS • Calorimetric
• Potentiometric
• Amperometric
• Optical
• Piezo-electric
• Immunosensors

What are biosensors?

• A biosensor is an analytical device
which converts a biological response
into an electrical signal

• sensor devices used in order to determine

the concentration of substances and other
parameters of biological interest

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Figure 6.1. Schematic diagram showing the
main components of a biosensor.
• The biocatalyst (a) converts the substrate to product.
• This reaction is determined by the transducer (b) which
converts it to an electrical signal.
• The output from the transducer is amplified (c),
processed (d) and displayed (e).
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Biosensors comprises of 2
components:
1. Biological sensing agent
enzymes, cells, DNA
2. Electronic transducers
changes/ converts a biological reaction into
a response/ signal that can be processed
and displayed
A successful biosensor must possess at least
some of the following beneficial features:
1. The biocatalyst must be highly specific for the purpose of the
analyses, be stable under normal storage conditions and, except
in the case of colorimetric enzyme strips and dipsticks, show good
stability over a large number of assays (i.e. much greater than
100).
2. The reaction should be as independent of such physical
parameters as stirring, pH and temperature as is manageable.
This would allow the analysis of samples with minimal pre-
treatment.
3. The response should be accurate, precise, reproducible and
linear over the useful analytical range, without dilution or
concentration. It should also be free from electrical noise.
4. If the biosensor is to be used for invasive monitoring in clinical
situations, the probe must be tiny and biocompatible, having no
toxic or antigenic effects. If it is to be used in fermenters it should
be sterilisable.
5. The complete biosensor should be cheap, small, portable and
capable of being used by semi-skilled operators.
6. There should be a market for the biosensor.
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Performance factors Time factors

• Selectivity – ability to discriminate between
different substances
• Sensitivity range (and dynamic range)
• Accuracy (usually <5%)
• Environmental condition (condition as pH,
temperature, ionic strength etc.)
• Response time
• Recovery time
• Working lifetime (and shelf time as well)
• Response time – time necessary to come
to an equilibrium and produce a reading
• Recovery time – time before the sensor is
ready for another measurement
• Lifetime:
– Degradation during continuous use
– Storage in wet condition
– Shelf life (in dry, in original packaging)

Areas of application Common assays in medicine

• Health care – measurements of blood,
other biological fluids, ions, metabolites to
show a patient metabolic state
• Industrial processes, e.g. Fermentation
• Environmental monitoring
• Food control
• Warfare detection

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electrode

Examples of common biosensors

• Glucose electrode
– Useful for diabetic patient Glucose oxidase +
catalase immobilised
– First biosensor ever created on electrode
Urine sample
containing glucose
Glucose
Glucose + O2 oxidase gluconic acid + H2O2
When enzymatic reactions H2O2 → O2 + 2e- + 2H+
catalase take place, oxygen will be
formed and detected by Gives of current reading
current reading due to
½ O2 + H 2 O reduction of oxygen at the Amount of glucose is proportional to
electrode current reading

Types of transducers 1. Optical biosensor

1. Optical – measure light intensity 1. determining changes in light absorption between the reactants
and products of a reaction
2. Amperometric – measure changes in current flow – colorimetric test strips, disposable single-use cellulose pads
when potential is constant impregnated with enzyme and reagents.
– The most common use is for whole-blood monitoring in diabetes
3. Potentiometric – measures changes in potential when control; the strips include glucose oxidase, horseradish peroxidase
current is constant (usually zero) and a chromogen. The hydrogen peroxide, produced by the aerobic
oxidation of glucose, oxidising the weakly coloured chromogen to a
4. Thermistors/calorimetric – measures heat of a highly coloured dye.
system
peroxidase
5. Piezoelectric – measures change in mass of chromogen(2H) + H2O2 → dye + 2H2O
adsorbed material
– evaluation of the dyed strips is best achieved by the use of portable
6. Conductiometric – measures the conc of a free reflectance meters, although direct visual comparison with a
charged ionic species btw 2 electrodes coloured chart is often used.

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2. measuring the light output by a luminescent process

• uses firefly luciferase to detect the presence of bacteria in food
or clinical samples.
• bacteria are specifically lysed and the ATP released (roughly
proportional to the number of bacteria present) reacted with D-
luciferin and oxygen in a reaction which produces yellow light
in high quantum yield
• luciferase
ATP + D-luciferin + O2 → oxyluciferin + AMP +
pyrophosphate + CO2 + light (562 nm)

• light produced may be detected photometrically by use of

high-voltage, and expensive, photomultiplier tubes or low-
voltage cheap photodiode systems

2. Amperometric biosensors
• function by the production of a current
when a potential is applied between two
electrodes.
• common usage involve the Clark oxygen
electrode

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Schematic diagram of a simple
amperometric biosensor
• A potential is applied between the
central platinum cathode and the
annular silver anode.
• This generates a current (I) which is
carried between the electrodes by
means of a saturated solution of KCl.
• This electrode compartment is
separated from the biocatalyst (here
shown glucose oxidase, GOD) by a
thin plastic membrane, permeable only
to oxygen.
• The analyte solution is separated from
the biocatalyst by another membrane,
permeable to the substrate(s) and
product(s).
• This biosensor is normally about 1 cm
in diameter but has been scaled down
to 0.25 mm diameter using a Pt wire
cathode within a silver plated steel
needle anode and utilising dip-coated
membranes.
3. Potentiometric biosensors
• make use of ion-selective electrodes in order to
transduce the biological reaction into an
electrical signal.
• consists of an immobilised enzyme membrane
surrounding the probe from a pH-meter
• The reaction occurring next to the thin sensing
glass membrane causes a change in pH which
may be read directly from the pH-meter's
display.
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Amperometric biosensors for flavo-
oxidase enzymes
• illustrating the three generations in
the development of a biosensor.
• The biocatalyst is shown
schematically by the cross-
hatching.
• (a) First generation electrode
utilising the H2O2 produced by the
reaction. (E0 = +0.68 V).
• (b) Second generation electrode
utilising a mediator (ferrocene) to
transfer the electrons, produced
by the reaction, to the electrode.
(E0 = +0.19 V).
• (c) Third generation electrode
directly utilising the electrons
produced by the reaction. (E0 =
+0.10 V). All electrode potentials
(E0) are relative to the Cl-
/AgCl,Ag0 electrode
A simple potentiometric biosensor
• A semi-permeable
membrane (a) surrounds
the biocatalyst (b)
entrapped next to the
active glass membrane
(c) of a pH probe (d).
• The electrical potential (e)
is generated between the
internal Ag/AgCl
electrode (f) bathed in
dilute HCl (g) and an
external reference
electrode (h).
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Potentiometric Biosensors:
Potentiometric Biosensors: pH linked
Ammonia linked
• Urea (in alkaline solution liberated ammonia can
be detected. The sensitivity up to 10-6M)

Potentiometric Biosensors: CO2 linked 4. Thermistors/calorimetric

• Many enzyme catalysed reactions are exothermic,
generating heat (Table 6.1) which may be used as a
basis for measuring the rate of reaction and, hence, the
analyte concentration.
• The temperature changes are usually determined by
means of thermistors at the entrance and exit of small
packed bed columns containing immobilised enzymes
within a constant temperature environment
• The thermistors, used to detect the temperature change,
function by changing their electrical resistance with the
temperature

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Table 6.1. Heat output (molar enthalpies) of

Schematic diagram of a enzyme catalysed reactions
calorimetric biosensor Heat output
• The sample stream (a) passes Reactant Enzyme
-H (kJ mole-1)
through the outer insulated box
(b) to the heat exchanger (c) Cholesterol Cholesterol oxidase 53
within an aluminium block (d).
• From there, it flows past the Esters Chymotrypsin 4 - 16
reference thermistor (e) and Glucose Glucose oxidase 80
into the packed bed bioreactor
(f, 1ml volume), containing the Hydrogen peroxide Catalase 100
biocatalyst, where the reaction
occurs. Penicillin G Penicillinase 67
• The change in temperature is Peptides Trypsin 10 - 30
determined by the thermistor
(g) and the solution passed to Starch Amylase 8
waste (h).
Sucrose Invertase 20
• External electronics (l)
determines the difference in Urea Urease 61
the resistance, and hence
temperature, between the Uric acid Uricase 49
thermistors.

5.Piezoelectric biosensors Immunosensors

• The phenomenon of piezoelectricity is used for mass
sensitive transducers.
• The material mostly used for piezoelectric biosensors is
quartz, cut at a special angle (AT-cut)
• Piezo-electric crystals (e.g. quartz) vibrate under the
influence of an electric field. The frequency of this
oscillation (f) depends on their thickness and cut, each
crystal having a characteristic resonant frequency.
• the change in frequency is proportional to the mass of
absorbed material, up to about a 2% change. This
frequency change is easily detected by relatively
unsophisticated electronic circuits.
• Biosensors may be used in conjunction with enzyme-linked
immunosorbent assays (ELISA) to form immunosensors, in order
to increase their range, speed and sensitivity
• ELISA is used to detect and amplify an antigen-antibody
reaction; the amount of enzyme-linked antigen bound to the
immobilised antibody being determined by the relative
concentration of the free and conjugated antigen and quantified
by the rate of enzymic reaction.
• Enzymes with high turnover numbers are used in order to achieve
rapid response.
• The sensitivity of such assays may be further enhanced by utilising
enzyme-catalysed reactions which give intrinsically greater
response; for instance, those giving rise to highly coloured,
fluorescent or bioluminescent products.

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Enzyme-linked immunosorbent The sandwich or double antibody technique

assay = ELISA….Basic principle
1. begins with an antibody bound to a
• a biochemical technique used mainly in immunology to detect polystyrene well plus the antigen to
the presence of an antibody or an antigen in a sample. be measured

• The ELISA has been used as a diagnostic tool in medicine 2. An enzyme conjugate is then
and plant pathology, as well as a quality control check in added to the well with bound
various industries. antigen-antibody or immune
complex
• used for antigen measurements.
3. Next a substrate is added to the
enzyme conjugate which is bound
• It is a heterogeneous, solid phase assay that requires the to the immune complex
separation of reagents.
4. From here if there are changes due
• ELISA has two available techniques for antigen to the presence of the enzyme
conjugate bound to the immune
measurement, the sandwich technique and the competitive complex, a positive test or color
technique. change will occur

Antigen-enzyme conjugate in excess Unlabeled antigen in excess

The antigen competitive inhibition assay
1. begins with an antibody bound to
a polystyrene well plus a test
sample containing an antigen
mixture to which an antigen-
enzyme conjugate is added.
2. At this point competitive inhibition
occurs between the antigen- • After the formation of an immune complex from an antigen-antibody
enzyme conjugate and an binding, the reagents are separated by a washing.
unlabeled antigen, depending on • Next a substrate is added to the immune complex. If the antigen-
which antigen type is in excess, enzyme conjugate is the antigen in excess a color change will occur
two different outcomes can follow indicating that the substrate was chemically changed as a result of the
when binding to a specific enzyme conjugate being bound to the immune complex.
antibody occurs. • If it is the unlabeled antigen that is in excess there will be little to no
change in color because the test sample contains antibody-type-
specific antigen.

Unlabeled antigen in excess of

Antigen-enzyme conjugate in excess
antigen-enzyme conjugate

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Immunosensors Immunological
Enzyme-Linked Immunosorbent Assay Response (ELISA)

ELISA is used to detect and amplify an antigen-antibody

reaction; the amount of enzyme-linked antigen bound to
the immobilised antibody is determined by the relative
concentration of the free and conjugated antigen and
quantified by the rate of enzymatic reaction.

There are two different principles

A
B

Antibody
enzyme
conjugate 1
antigen

(B) After a suitable period unbound material is

(A)A tube is coated with antigen. An excess of
washed off.
specific antibody-enzyme conjugate is placed in
the tube and allowed to bind.

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(A) A transducer is coated with (immobilised)

antibody, specific for the antigen of interest. The
transducer is immersed in a solution containing a
mixture of a known amount of antigen-enzyme
conjugate plus unknown concentration of sample
antigen.

Antigen-enzyme A
(C) The analyte antigen solution is passed into the conjugate
Antigen
tube, binding and releasing some of the antibody-
enzyme conjugate dependent upon the antigen's 2
concentration. antibody

(D) Amount of antibody-enzyme conjugate released

is measured by the response from the biosensor. transducer

(B) After suitable period the antigen and antigen- (C) Unbound material is washed off. The amount
enzyme conjugate will be distributed between the of antigen-enzyme conjugate bound is determined
bound and free states dependent upon their relative from the transduced signal.
concentrations.

B C

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The end

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