SENSORS

by

Luciana V. Ilao

Associate Professor in Chemistry

Department of Physical Sciences and Mathematics
University of the Philippines Manila

1
What is a sensor?

It is a device that detects or measures a physical

property and records, indicates or otherwise
responds to it.

Types of sensors
A. physical sensors: for measuring distance, mass,
temperature, pressure, etc.;
B. chemical sensors: measure chemical substances
by chemical or physical responses; and
C. biosensors which measure chemical substances
by using a biological sensing element.

2
Biosensors
A biosensor is an analytical device which
converts a biological response into an electrical
signal (Figure 1).

The term 'biosensor' is often used to cover

sensor devices used in order to determine the
concentration of substances and other
parameters of biological interest even where
they do not utilize a biological system directly.

3
Figure 1. Schematic of a Biosensor

4
Analogy with the nose as a
sensor (actually a biosensor),

Olfactory membrane - biological recognition

element
Nerve cell - transducer,
Nerve fibre - actuator, i.e., the device that produces
the display
Brain - measuring element.

*Eggins, B. R., Biosensors: An Introduction,

Copyright
1996. John Wiley & Sons Limited.

5
Figure 2. 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). 6
Aspects of Sensors
Recognition Element
• Key component of any sensor device.
• Impart the selectivity that enables the sensor
to respond selectively to a particular analyte or
group of analytes, thus avoiding interferences
from other substances.
• In biosensors, the most common recognition
element is an enzyme. Others include antibodies,
nucleic acids and receptors.

7
 Biosensors/Biochips

Bioreceptor Transducer

Antibody DNA Biomimetic Optical Mass–based

Enzyme Cell/Tissue Electrochemical Other

Cellular Non–enzymatic
Systems proteins

Figure 3. Biosensor/Biochip Classification Scheme

8
 (Recognition Element)

A. Biocatalytic Recognition Element

☛ The biosensor is based on a reaction catalysed by
macromolecules, which can be:
✔present in their original biological environment;
✔have been isolated previously; or
✔have been manufactured.
☛ A continuous consumption of substrate(s) is
achieved by the immobilized biocatalyst
incorporated into the sensor.
☛ Transient or steady-state responses are monitored
by the integrated detector.

9
 Recognition
Element
Types of biocatalyst commonly used:
A. Enzyme (mono-or multi-enzyme): the most
common and well-developed recognition system;
B. Whole cells (micro-organisms, such as bacteria,
fungi, eukaryotic cells or yeast) or cell organelles or
particles (mitochondria, cell walls);
C. Tissue (plant or animal tissue slice).

❚ One or more analytes react in the presence of

enzyme(s), whole cells or tissue culture and yield
one or several products according to the general
reaction scheme:
biocatalyst
S + S0 → P + P0

10
 (Recognition Element)

Strategies that use adjacent transducers for

monitoring the analyte consumption by
biocatalysed reaction:

1. detection of the co-substrate (S0) consumption,

e.g. oxygen depleted by oxidase, bacteria or
yeast reacting layers, and the corresponding
signal decrease from its initial value;
2. recycling of one of the reaction products (P),
e.g. hydrogen peroxide, H, CO2, NH3, etc.,
production by oxidoreductase, hydrolase,
lyase, etc., and corresponding signal increase;

11
 (Recognition Element)
Strategies that use adjacent transducers

3. detection of the state of the biocatalyst redox active

centre, cofactor, prosthetic group evolution in the
presence of substrate S, using an immobilized
mediator which reacts sufficiently rapidly with the
biocatalyst and is easily detected by the transducer;
e.g. various ferrocene derivatives, as well as
tetrathiafulvalene-tetracyanoquinodimethane (TTF+;
TCNQ-) organic salt, quinones, quinoid dyes, Ru or Os
complexes in a polymer matrix
4. direct electron transfer between the active site of a
redox enzyme and the electrochemical transducer.

12
 (Recognition Element)
Strategies for improving biosensor performance
when several enzymes are immobilized within
the same reaction layer:
1. Several enzymes facilitate the biological recognition
☛ sequentially converting the product of a series of enzymatic
reactions into a final electroactive form
☛ set-up allows a much wider range of possible biosensor
analytes;
2. Multiple enzymes, applied in series
☛ may regenerate the first enzyme co-substrate
☛ a real amplification of the biosensor output signal may be
achieved by efficient regeneration of another co-substrate
of the first enzyme.

13
 (Recognition Element)
Strategies for improving biosensor performance (cont’d)

3. multiple enzymes, applied in parallel

☛ may improve the biosensor selectivity by
decreasing the local concentration of
electrochemical interfering substance
☛ an alternative to the use of either a
permselective membrane or a differential set-
up, i.e. subtraction of the output signal
generated by the biosensor and by a reference
sensor having no biological recognition
element

14
Biocomplexing or bioaffinity recognition element

☛ biosensor operation is based on the interaction of the

analyte with macromolecules or organized molecular
assemblies that have either been isolated from their
original biological environment or engineered;
☛ Equilibrium is usually reached and there is no further net
consumption of the analyte by the immobilized
biocomplexing agent.
☛ Equilibrium responses are monitored by the integrated
detector.
☛ In some cases, this biocomplexing reaction is monitored
using a complementary biocatalytic reaction.
☛ Steady-state or transient signals are then monitored by the
integrated detector.

15
Antibody–antigen interaction
The most developed examples of biosensors using
biocomplexing receptors are based on
immunochemical reactions, i.e. binding of the
antigen (Ag) to a specific antibody (Ab).

Formation of such Ab±Ag complexes has to be

detected under conditions where non-specific
interactions are minimized.

Each Ag determination requires the production of a

particular Ab, its isolation and, usually, its
purification.

16
Aspects of Sensors (cont’d)

Transducer – the detector device (shown as the 'black

box' in Figure 2) which makes use of a physical change
accompanying the reaction.

A. Electrochemical Transducers
1. Potentiometric.
• Based on changes in the distribution of charges
causing an electrical potential to be produced
(potentiometric biosensors)
• Involve the measurement of the emf (potential) of a
cell at zero current.
• The emf is proportional to the logarithm of the
concentration of the substance being determined.

17
Aspects of Sensors (cont’d)

2. Voltammetric
• Based on the movement of electrons produced in a
redox reaction
• An increasing (decreasing) potential is applied to the
cell until oxidation (reduction) of the substance to be
analyzed occurs and there is a sharp rise (fall) in the
current to give a peak current.
• The height of the peak current is directly proportional
to the concentration of the electroactive material.
• If the appropriate oxidation (reduction) potential is
known the current may be observed at the potential.
This mode is known as amperometric.

18
Aspects of Sensors (cont’d)

3. Conductometric. Involves measurement of

change in electrical conductivity that results
from the composition of the solution.
4. FET-based sensors. Electrochemical
transducers on a silicon chip-based field-
effect transistor.
• Mainly been used with potentiometric
sensors, but could also be used with
voltammetric or conductometric sensors.

19
Aspects of Sensors (cont’d)

B. Optical Transducers
• Based on the light output during the reaction
or a light absorbance difference between the
reactants and products
• include absorption spectroscopy, fluorescence
spectroscopy, luminescence spectroscopy,
internal reflection spectroscopy, surface
plasmon spectroscopy and light scattering.

20
Aspects of Sensors (cont’d)

C. Piezo-electric Devices
• Based on effects due to the mass of the
reactants or products (piezo-electric
biosensors).
• Devices that involve the generation of
electric currents from a vibrating crystal.
• The frequency of vibration is affected by the
mass of material adsorbed on its surface,
which could be related to changes in a
reaction.
• Includes surface acoustic wave devices

21
Aspects of Sensors (cont’d)

D. Thermal Sensors
• Based on the production or absorption of heat
during chemical and biochemical processes.
• The heat can be measured by sensitive
thermistors and hence be related to the
amount of substance to be analyzed.

22
Fig. 3. Configuration of a biosensor showing
biorecognition, interface, and transduction
elements.
23
 Table 1. Biosensor Components
Transducer Measurement Typical
System Mode Applications

Ion-Selective Potentiometric Ions in biological

Electrode media, enzyme
electrodes

Gas-Sensing Potentiometric Gases, enzyme,

Electrodes organelle, cell or
tissue electrodes

Field-Effect Potentiometric Ions, gases,

Transistors enzyme substrates
immunological
analytes

24
Optoelectronic Optical pH; enzymes;
and Fiber–Optic immunological
Devices analytes

Thermistors Calorimetric Enzyme, organelle,

gases, pollutants,
antibiotics, vitamins
Enzyme Electrodes Amperometric Enzymes, immunological
systems

Conductimetric Conductance Enzyme substrates

Piezoelectric Acoustic (mass) Volatile gases and
Crystals vapors, antibodies

25
Beneficial features of a Biosensor:

1. Biocatalyst
❚ highly specific for the purpose of the
analyses,
❚ stable under normal storage conditions, and
❚ show good stability over a large number of
assays, i.e., much greater than 100 (except
in the case of colorimetric enzyme strips and
dipsticks)

26
Beneficial features of a Biosensor(cont’d):

2. Reaction
❚ should be as independent of physical parameters
such as stirring, pH and temperature as is
manageable for minimal samples pre-treatment

3. Response
❚ determined by the biocatalytic membrane which
accomplishes the conversion of reactant to product
❚ should be accurate, precise, reproducible and linear
over the useful analytical range, without dilution or
concentration
❚ should be free from electrical noise.

27
Beneficial features of a Biosensor (cont’d):

4. The probe must be:

❚ tiny and biocompatible, having no toxic or antigenic
effects
❚ should be sterilisable (preferably by autoclaving but
no biosensor enzymes can presently withstand such
drastic wet-heat treatment)
❚ should not be prone to fouling or proteolysis.
❚ be cheap, small, portable and capable of being used
by semi-skilled operators.

5. There should be a market for the biosensor.

28
Immobilization of Enzymes

♦ The biorecognition element has to be properly

attached to the transducer to make a functional
biosensor

Qualities of a Good Immobilization Technique:

1. It should not reduce the activity or the specificity
of the biorecognition element.
2. The stability of the element should be
maintained or increased in order to make the
biosensor reusable.
3. The method should be easily repeatable so that
large scale production is possible.

29
Principal methods for enzyme
immobilization
a. Adsorption to the surface
◆ can either be physical or chemical
◆ generally used only for short term
applications because it is a relatively simple
process that requires no reagents making it
easy to setup
◆ Disadvantage: Not easily reproduced and the
resulting biosensor does not keep its
properties over time(Diamond; Eggins).

30
• Physical adsorption

* can take place on solid surfaces such as

alumina, charcoal, clay, cellulose, silica gel,
glass, and collagen.
* Uses hydrogen bonds, van der Waals forces,
salt linkages, and hydrophobic interactions.
* The resulting bonds are not stable and are
susceptible to changes in pH, temperature,
and ionic strength

31
• Chemical adsorption
* Utilizes a nucleophilic group to couple the
biorecognition material to the transducer or
membrane.
* The resulting coupling bonds are typically
covalent bonds.
* The nucleophilic group is specifically chosen
so that it does not contribute to the overall
catalytic activity of the biorecognition
element.
* Common nucleophilic groups used: NH2, CO2H,
OH, C6H4OH, SH, and the heterocyclic
imidazole..

32
b. Covalent binding: Covalent chemical bonds are
formed between the selective component and
the transducer.
* Cross-linking, a multifunctional crosslinking
reagent is used to bind the biomaterial to solid
supports on the transducer surface.
* Often used in the stabilization of adsorbed
proteins, where a reagent is used to link inert
proteins together.
* Adds greater stability to the system.
* Generally damage to the enzyme and has poor
mechanical strength.
* Should only be used in short term application.

33
covalent binding(cont’d)

* Should not be used when antibodies are the

biorecognition element because it can orient
the antibody binding site so that antibody-
antigen binding will not be favored and will
result in an inaccurate sensor (Diamond;
Eggins).
*Common molecules used for cross-linking
(Eggins).

34
Enzyme Immobilization (cont’d)

c. Entrapment
* Where the selective element is physically
entrapped in a matrix of a gel, paste or polymer
* Typical materials used for this outer gel matrix
are polyacrylamide, polyvinyl alcohol, polyvinyl
chloride, epoxy, sol-gel processed glass, or a
Langmuir-Blodgett film (Prasad).

35
d. Membrane confinement (Microencapsulation)
* Trapping between membranes – one of the earliest
methods to be employed.
* Membranes with varying porosities are used to
entrap the biorecognition material close to the
transducer surface.
* Keeps the biorecognition material close to the
transducer while not actually immobilizing the
material.
* Tends to be very reliable and adaptable.
* Disadvantage: Diffusional resistance through the
membrane which can be used as an advantage by
reducing the pore size so only molecules of the
target analyte can pass through the membrane.

36
Fig.5. The four principal methods for enzyme immobilization

37
 Advantageous features of immobilized enzymes:

1. They may be re-used, which ensures that the

same catalytic activity is present for a series of
analyses. This is an important factor in securing
reproducible results and avoids the pitfalls
associated with the replicate pipetting of free
enzyme otherwise necessary in analytical
protocols.

2. Many are intrinsically stabilized by the

immobilization process

38
Advantageous features of immobilized enzymes(cont’d):

3. An excess of enzyme within the immobilised

sensor system gives a catalytic redundancy (i.e.
h << 1) that is sufficient to ensure an increase
in the apparent stabilization of the immobilized
enzyme.

4. Lower cost relative to the analytical usage of free

soluble enzymes.

39
Performance Factors of a Biosensor

1. Selectivity.
• The most important characteristic of sensors
• The ability to discriminate between different substances.
• Principally a function of the selective component, although sometimes
the operation of the transducer contributes to the selectivity.
2. Sensitivity range.
• The change in response per unit change in concentration of analyte
• Usually needs to be sub-millimolar, but in special cases can go down to
the femtomolar (10-15 M) range.
3. Accuracy. This needs to be better than ±5%.

40
Performance Factors (cont’d)

4. Nature of solution. Conditions such as pH,

temperature and ionic strength.
5. Response time. Usually much longer (30 s or
more) with biosensors than with chemical
sensors.
6. Recovery time. The time that elapses before the
sensor is ready to analyze the next sample – it
must not be more than a few minutes.
7. Working lifetime is usually determined by the
stability of the selective material. For biological
materials this can be a short as a few days,
although it is often several months or more.

41
Generations of biosensors (Fig.4):

First generation biosensors: the normal product

of the reaction diffuses to the transducer and
causes the electrical response;

Second generation biosensors: involve specific

'mediators' between the reaction and the
transducer in order to generate improved
response; and

Third generation biosensors: the reaction itself

causes the response and no product or
mediator diffusion is directly involved.

42
Fig.4. 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.

All electrode potentials (E0) are relative to the

Cl-/AgCl,Ag0 electrode.

The following reaction occurs at the enzyme in all

three biosensors:

Substrate(2H) + FAD-oxidase → Product + FADH2-

oxidase

43
biocatalyst:
FADH2-oxidase + O2 → FAD-oxidase + H2O2

electrode: H2O2 → O2 + 2H+ + 2e-

Fig.4a. First generation electrode utilising the H2O2

produced by the reaction. (E0=+0.68 V).
44
Biocatalyst:
FADH2-oxidase + 2 Ferricinium+ → FAD-oxidase + 2
Ferrocene + 2H+
electrode : 2 Ferrocene → 2 Ferricinium+ + 2e-

Fig 4b. Second generation electrode utilising a mediator

(ferrocene) to transfer the electrons, produced by the
reaction, to the electrode. (E0= +0.19 V).
45
 biocatalyst/electrode
FADH2-oxidase → FAD-oxidase + 2H+ + 2e-

Fig 4c.Third generation electrode directly utilising the electrons

produced by the reaction. (E0= +0.10 V).
46
Calorimetric Biosensors

❚ The most generally applicable type of

biosensor
❚ based on heat evolved by exothermic
reactions (Table 2).
❚ may be used as a basis for measuring the rate
of reaction and the analyte concentration.
❚ 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 (Figure 5).

47
Calorimetric Biosensors

❚ Registers up to 80% of the heat generated in the

reaction under closely controlled conditions as a
temperature change in the sample stream.
❚ Can cause a change in temperature of the solution
amounting to approximately 0.02°C (the temperature
change commonly encountered)
❚ Necessitates a temperature resolution of 0.0001°C for
the biosensor to be generally useful.

48
Table 2. Molar enthalpies of enzyme catalyzed reactions.
-1
Reactant Enzyme ∆H (kJ mole )
Cholesterol Cholesterol oxidase – 53
Esters Chymotrypsin – 4 – 16
Glucose Glucose oxidase – 80
Hydrogen peroxide Catalase – 100
Penicillin G Penicillinase – 67
Peptides Trypsin – 10 - 30
Starch Amylase –8
Sucrose Invertase – 20
Urea Urease – 61
Uric acid Uricase – 49

49
Figure 5. Schematic diagram of a
calorimetric biosensor. The sample
stream (a) passes through the outer
insulated box (b) to the heat
exchanger (c) within an aluminium
block (d). From there, it flows past
the reference thermistor (e) and
into the packed bed bioreactor (f,
1mL volume) containing the
biocatalyst, where the reaction
occurs. The change in temperature
is determined by the thermistor (g)
and the solution passed to waste
(h). External electronics (l)
determines the difference in the
resistance, and hence temperature,
between the thermistors.
50
The thermistors used to detect the temperature
change, function by changing their electrical
resistance with the temperature, obeying the
relationship

ln(R1/R2)=B(1/T1 - 1/T2)
therefore:
(R1/R2)=e(B(1/T1–1/T2))

where:
R1 and R2 are the resistances of the thermistors at
absolute temperatures T1 and T2 respectively; and
B is a characteristic temperature constant for the
thermistor.

51
When the temperature change is very small

B(1/T1) – (1/T2) << 1

the relationship may be simplified by the approximation

when x<<1 that ex ≈ 1 + x (where x is B(1/T1) – (1/T2),

R1=R2 {1 + B [(T2 – T1) / T1T2]}

As T1 ≈ T2, they both may be replaced in the denominator by T1.

∆ R/R = – (B/T12)∆ T
The relative decrease in the electrical resistance (∆ R/R) of the
thermistor is proportional to the increase in temperature (∆ T).

52
The sensitivity (10-4 M) and range (10-4 - 10-2 M) of thermistor
biosensors are both quite low for the majority of applications
although greater sensitivity is possible using the more
exothermic reactions (e.g. catalase).

The low sensitivity of the system can be increased substantially

by increasing the heat output by the reaction. This is achieved
by linking together several reactions in a reaction pathway, all
of which contribute to the heat output.

Ex. The sensitivity of glucose analysis using glucose oxidase can

be more than doubled by co-immobilisation of catalase within
the column reactor in order to disproportionate the hydrogen
peroxide produced. An extreme case of this amplification is
shown in the following recycle scheme for the detection of
ADP.

53
54
Potentiometric Biosensors

❚ These 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 (Figure
4), where the catalysed reaction generates or
absorbs hydrogen ions (Table 3).
❚ 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.

55
Figure 6. 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).

56
Types of ion-selective electrodes which are of
use in biosensors:

1. Glass electrodes for cations (e.g. normal pH

electrodes) in which the sensing element is a very
thin hydrated glass membrane that generates a
transverse electrical potential due to the
concentration-dependent competition between the
cations for specific binding sites.

❚ The selectivity of this membrane is determined by

the composition of the glass. The sensitivity to H+
is greater than that achievable for NH4+

57
Types of ion-selective electrodes which are of
use in biosensors:
2. Glass pH electrodes coated with a gas-permeable
membrane selective for CO2, NH3 or H2S.
❚ The diffusion of the gas through this membrane causes a
change in pH of a sensing solution between the
membrane and the electrode which is then determined.

3. Solid-state electrodes where the glass membrane is

replaced by a thin membrane of a specific ion conductor
made from a mixture of silver sulfide and a silver halide.
❚ The iodide electrode is useful for the determination of I-
in the peroxidase reaction (Table 3c) and also responds
to cyanide ions.

58
Table 3. Reactions involving the release or absorption of ions
that may be utilised by potentiometric biosensors.
(a) H+ cation
glucose oxidase H2O
D–glucose + O2 → D–glucono–1,5–lactone + H2O2 → D–gluconate + H+
Penicillinase
Penicillin → penicilloic acid + H+

urease (pH 6.0)a

H2NCONH2 + H2O + 2H+ → 2NH4+ + CO2

urease (pH 9.5)b

H2NCONH2 + 2H2O → 2NH3 + HCO3– + H+

Lipase
neutral lipids + H2O → glycerol + fatty acids + H+
a
Can also be used in NH4+ and CO2(gas) potentiometric biosensors.
b
Can also be used in an NH3(gas) potentiometric biosensor

59
Table 3. Reactions involving the release or absorption of ions that
may be utilised by potentiometric biosensors(cont’d).
(b) NH4+ cation
L–amino acid oxidase
L–amino acid + O2 + H2O → keto acid + NH4+ + H2O2
Asparaginase
L–asparagine + H2O → L–aspartate + NH4+
urease (pH 7.5)
H2NCONH2 + 2H2O + H+ → 2NH4+ + HCO3–
(c) I– anion
Peroxidase
H2O2 + 2H+ + 2I– → I2 + 2H2O
(d) CN-anion,
β –glucosidase
amygdalin + 2H2O → 2glucose + benzaldehyde + H+ + CN–

60
 The response of an ion–selective electrode is given by
ε = ε ° + RT (ln[i]/zF)
where:
ε = the measured potential (in volts)
ε ° = characteristic standard potential for the ion–
selective/external electrode system
R = the gas constant
T = absolute temperature (K)
z = the signed ionic charge
F = Faraday’s constant
[i] = concentration of free uncomplexed ionic species
(strictly, [i] should be the activity of the ion but at the concentrations normally
encountered in biosensors, this is effectively equal to the concentration).

61
• This means, for example, that there is an increase in
the electrical potential of 59 mv for every decade
increase in the concentration of H+ at 25°C.

• The logarithmic dependence of the potential on the

ionic concentration is responsible both for the wide
analytical range and the low accuracy and precision of
these sensors.

• Their normal range of detection is 10–4 – 10–2 M, a

minority are ten–fold more sensitive.

• Typical response time are between one and five

minutes allowing up to 30 analyses every hour.

62
❚ Biosensors that involve H+ release or utilisation
necessitate the use of very weakly buffered
solutions (i.e. < 5 mM) if a significant change in
potential is to be determined.

❚ The relationship between pH change and substrate

concentration is complex, including other such non-
linear effects as pH-activity variation and protein
buffering.

❚ However, conditions can often be found where there

is a linear relationship between the apparent change
in pH and the substrate concentration.

63
• A recent development from ion–selective electrodes is
the production of ion–selective field effect transistors
(ISFETs) and their biosensor use as enzyme–linked
field effect transistors (ENFETs, Fig. 7).

• Enzyme membranes are coated on the ion–selective

gates of these electronic devices; the biosensor
responds to the electrical potential change via the
current output.

• These are potentiometric devices although they

directly produce changes in the electric current.

64
•Main advantage of ENFETS: their extremely small size
(<< 0.1 mm2) which allows cheap mass–produced
fabrication using integrated circuit technology.

•As an example, a urea-sensitive FET (ENFET containing

bound urease with a reference electrode containing bound
glycine) has been shown to show only a 15% variation in
response to urea (0.05 - 10.0 mg ml-1) during its active
lifetime of a month.

•Several analytes may be determined by miniaturised

biosensors containing arrays of ISFETs and ENFETs.

•The sensitivity of FETs, however, may be affected by the

composition, ionic strength and concentrations of the
solutions analysed.

65
 Fig.7. Schematic diagram
of the section across the
width of an ENFET. The
actual dimensions of the
active area is about 500
mm long by 50 mm wide
by 300 mm thick. The main
body of the biosensor is a
p-type silicon chip with two
n-type silicon areas; the
negative source and the
positive drain. The chip is
(0.1 mm thick) of silica (SiO2) which forms theinsulated
gate of the FET.
by a thinAbove
layer this
gate is an equally thin layer of H+ -sensitive material (e.g. tantalum
oxide), a protective ion selective membrane, the biocatalyst and the
analyte solution, which is separated from sensitive parts of the FET by an
inert encapsulating polyimide photopolymer. When a potential is applied
between the electrodes, a current flows through the FET dependent upon
the positive potential detected at the ion-selective gate and its
consequent attraction of electrons into the depletion layer. This current (I)
is compared with that from a similar, but non-catalytic ISFET immersed in
the same solution. (Note that the electric current is, by convention, in the
opposite direction to the flow of electrons). 66
Amperometric Biosensors
• These biosensors function by the production of a current
when a potential is applied between two electrodes.

• They generally have response times, dynamic ranges and

sensitivities similar to the potentiometric biosensors.

• The simplest amperometric biosensors in common usage

involve the Clark oxygen electrode

67
The Response of an Amperometric Biosensor

The current (i) produced by such amperometric biosensors is

related to the rate of reaction (vA) by the expression:

i = nFAvA
where:
n = the number of electrons transferred
A = electrode area; and
F = Faraday’s constant.

Usually the rate of reaction is made diffusionally controlled by use of

external membranes. Under these circumstances the electric current
produced is proportional to the analyte concentration and independent
both of the enzyme and electrochemical kinetics.

68
Fig. 8. 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 utilizing dip-coated
membranes. 69
Methods of Optical Transduction

Properties of optical signal produced by the

biorecognition of an analyte:
1. Phase change: Happens when the real part of the
biorecognition material’s refractive index changes.
* Shown as a polarization change of the input light
source, a change in the propagation characteristics,
or a change in the optical field distribution.
2. Amplitude changes: Caused by absorption or
reflection of the input light.
* Shown as a loss of intensity in the output light as
compared to the input source.

70
Methods of Optical Transduction(cont’d)

3. Frequency changes of the output light: Can be

measured using a fluorescent dye that fluoresces
in the presence of an analyte with a frequency
that is Stokes-shifted away from the original
sensing light.
* Raman scattering can also be used which also
uses the same principle of a Stokes-shifted
frequency but it is due to vibrational excitation.
4. Frequency shift: Can be caused by a nonlinear
optical interaction such as second-harmonic
generation in which the output beam is doubled
from what the input beams were.

71
Types of optical biosensors

1. fiber optic
2. planar waveguide
3. evanescent wave
4. interferometric
5. Surface plasmon resonance

72
Diagram of a fiber based optical biosensor Wolfbeis).

73
Main Areas of Development
1. Involves determining changes in light absorption between the reactants
and products of a reaction;
• Usually involve the widely established, if rather low technology, use of
colorimetric test strips which are disposable single–use cellulose pads
impregnated with enzyme and reagents.
• The most common use of this technology is for whole–blood monitoring
in diabetes control where the strips include glucose oxidase, horseradish
peroxidase (EC 1.11.1.7) and a chromogen (e.g. o-toluidine or 3,3',5,5'-
tetramethylbenzidine). The hydrogen peroxide, produced by the aerobic
oxidation of glucose oxidising the weakly coloured chromogen to a
highly coloured dye.
peroxidase
chromogen(2H) + H2O2 → dye + 2H2O
• The dyed strips are evaluated by the use of portable reflectance meters
or direct visual comparison with a colored chart.

74
2. Involves measuring the light output by a luminescent process.

• A biosensor involving luminescence uses firefly luciferase (Photinus–

luciferin 4–monooxygenase (ATP–hydrolysing) 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) react with D–
luciferin and oxygen in a reaction that produces yellow light in high
quantum yield.
luciferase
ATP + D–luciferin + O2 → oxyluciferin + AMP + pyrophosphate +
CO2 + light (562 nm)
• The light produced may be detected photometrically by use of high-
voltage and expensive photomultiplier tubes or low–voltage cheap
photodiode systems.

75
 Piezo-electric Biosensors
• 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.
• This resonant frequency changes as molecules adsorb or
desorb from the surface of the crystal, obeying the
relationships
∆ f = Kf 2 ∆ m/A
where
∆ f = the change in resonant frequency (Hz)
∆ m = change in mass of adsorbed material (g)
K = constant for the particular crystal dependent on such
factors as its density and cut, and
A = the adsorbing surface area (cm2).
76
antigen of interest is
immobilised on the surface
of a tube. A mixture of a
known amount of antigen-
enzyme conjugate plus
unknown concentration of
sample antigen is placed in
the tube and allowed to
equilibrate. (ii) After a
suitable period the antigen
and antigen-enzyme
conjugate will be
distributed between the
bound and free states
dependent upon their
relative concentrations. (iii)
Unbound material is
washed off and discarded.
The amount of antigen-
enzyme conjugate that is
bound may be determined
by the rate of the
subsequent enzymatic
reaction.
77
Principles of immunosensors. (a)(i) A tube is coated with (immobilised) antigen. An
excess of specific antibody-enzyme conjugate is placed in the tube and allowed to
bind. (a)(ii) After a suitable period any unbound material is washed off. (a)(iii) The
analyte antigen solution is passed into the tube, binding and releasing some of the
antibody-enzyme conjugate dependent upon the antigen's concentration. The amount
of antibody-enzyme conjugate released is determined by the response from the
biosensor.

78
(b) (i) 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. (ii) After a suitable
period the antigen and antigen-enzyme conjugate will be distributed
between the bound and free states dependent upon their relative
concentrations. (b)(iii) Unbound material is washed off and discarded.
The amount of antigen-enzyme conjugate bound is determined directly
from the transduced signal. 79
Schematic diagram of an immunosensor device.

80
Apparatus for piezoelectric sensor
81
What are Molecularly Imprinted Polymers
(MIPs)?

MIPs are polymers that contain highly selective

recognition sites as a result of polymerization
around a template molecule bound covalently or
non-covalently to functional monomers
To date a wide range of templates have been used
including organic compounds, sugars, peptides,
nucleotides, proteins, crystals and even cells

82
MIP synthesis is a three step process:
1. Assembly of the template with funtional
monomer units via covalent bonding, hydrogen
bonding,
hydrophobic, or ionic interactions;
2. Polymerization around the template/monomer
complex, incroporating the monomers into the
polymer
backbone to create a recognition pocket;
3. Removal of the template by washing with
organic solvent or acid/base hydrolysis.

83
 Uses of MIPs
1. Industrial Use
☛ Purification: selectively removing reaction
products or by-products
2. Organic Chemistry
☛ Stoichiometric Reagents: scavengers, passive
supports, supported reagents, protecting groups
☛ Catalysts: C-C bond formation, elimination
reactions, mimics of natural enzymes, transition
metal mediated reactions

84
Electron Microscopy
What are Electron Microscopes?

Electron Microscopes are scientific instruments that

use a beam of highly energetic electrons to
examine objects on a very fine scale. This
examination can yield the following information:

Topography
The surface features of an object or "how it looks",
its texture; direct relation between these features
and materials properties (hardness,
reflectivity...etc.)
85
Morphology: The shape and size of the particles making up
the object; direct relation between these structures and
materials properties (ductility, strength, reactivity...etc.)

Composition: The elements and compounds that the object

is composed of and the relative amounts of them; direct
relationship between composition and materials properties
(melting point, reactivity, hardness...etc.)

Crystallographic Information: How the atoms are arranged in

the object; direct relation between these arrangements
and materials properties (conductivity, electrical
properties, strength...etc.)

86
How do Electron Microscopes Work?

Electron Microscopes(EMs) function exactly as their

optical counterparts except that they use a
focused beam of electrons instead of light to
"image" the specimen and gain information as to
its structure and composition.

87
Basic steps involved in all EMs:
1. A stream of electrons is formed (by the Electron
Source) and accelerated toward the specimen using a
positive electrical potential.
2. This stream is confined and focused using metal
apertures and magnetic lenses into a thin, focused,
monochromatic beam.
3. This beam is focused onto the sample using a
magnetic lens.
4. Interactions occur inside the irradiated sample,
affecting the electron beam.
❚ These interactions and effects are detected and
transformed into an image

88
SEMs are patterned after Reflecting Light
Microscopes and yield similar information:

Topography: The surface features of an object or

"how it looks", its texture; detectable features
limited to a few manometers.

Morphology: The shape, size and arrangement of

the particles making up the object that are lying
on the surface of the sample or have been
exposed by grinding or chemical etching;
detectable features limited to a few manometers.

89
Composition: The elements and compounds the
sample is composed of and their relative ratios,
in areas ~ 1 micrometer in diameter.

Crystallographic Information: The arrangement of

atoms in the specimen and their degree of order;
only useful on single-crystal particles >20
micrometers.

90
A detailed explanation of how a typical SEM
functions follows (refer to the diagram below):

91
1. The "Virtual Source" at the top represents the electron
gun, producing a stream of monochromatic electrons.
2. The stream is condensed by the first condenser lens
(usually controlled by the "coarse probe current knob").
This lens is used to both form the beam and limit the
amount of current in the beam. It works in conjunction
with the condenser aperture to eliminate the high-angle
electrons from the beam.
3. The beam is then constricted by the condenser aperture
(usually not user selectable), eliminating some high-angle
electrons.

92
4. The second condenser lens forms the electrons into
a thin, tight, coherent beam and is usually
controlled by the "fine probe current knob".
5. A user selectable objective aperture further
eliminates high-angle electrons from the beam.
6. A set of coils then "scan" or "sweep" the beam in a
grid fashion (like a television), dwelling on points for
a period of time determined by the scan speed
(usually in the microsecond range).
7. The final lens, the Objective, focuses the scanning
beam onto the part of the specimen desired.

93
8. When the beam strikes the sample (and dwells for a
few microseconds) interactions occur inside the
sample and are detected with various instruments.
9. Before the beam moves to its next dwell point these
instruments count the number of interactions and
display a pixel on a CRT whose intensity is
determined by this number (the more reactions the
brighter the pixel).
10. This process is repeated until the grid scan is
finished and then repeated, the entire pattern can be
scanned 30 times per second.

94
Mechanically Alloyed Cu-Nb-Fe (500x)

95
Organically Deposited Iron Oxide (10,000x)

96
Scanning Electron Microscopy (SEM)
The scanning electron microscope (SEM)
☛ uses a focused beam of high-energy electrons to
generate a variety of signals at the surface of solid
specimens.
☛ The signals that derive from electron-sample
interactions reveal information about the sample such
as
✔external morphology (texture),
✔chemical composition; and
✔crystalline structure and orientation of materials
making up the sample.

97
Fundamental Principles of Scanning
Electron Microscopy (SEM)
☛ Accelerated electrons in an SEM carry significant
amounts of kinetic energy
☛ This energy is dissipated as a variety of signals produced
by electron-sample interactions when the incident
electrons are decelerated in the solid sample.
✔secondary electrons (that produce SEM images),
✔backscattered electrons (BSE),
✔diffracted backscattered electrons photons
(characteristic X-rays that are used for elemental
analysis and continuum X-rays),
✔visible light (cathodoluminescence--CL); and
✔heat.

98
Information Derived
☛ Secondary electrons
✔ for showing morphology and topography on samples
☛ backscattered electrons
✔ for illustrating contrasts in composition in multiphase samples
(i.e. for rapid phase discrimination)
☛ X-ray generation
✔ produced by inelastic collisions of the incident electrons with
electrons in discrete ortitals (shells) of atoms in the sample.
✔ As the excited electrons return to lower energy states, they yield
characteristic X-rays of a fixed wavelength (that is related to the
difference in energy levels of electrons in different shells for a
given element).

99
Transmission Electron Microscope (TEM)
TEMs are patterned after Transmission Light
Microscopes and will yield similar information.

Morphology: The size, shape and arrangement of

the particles which make up the specimen as well
as their relationship to each other on the scale of
atomic diameters.

Crystallographic Information: The arrangement of

atoms in the specimen and their degree of order,
detection of atomic-scale defects in areas a few
nanometers in diameter

100
Compositional Information (if so equipped): The elements
and compounds the sample is composed of and their
relative ratios, in areas a few nanometers in diameter

A TEM works much like a slide projector.

✔ A projector shines a beam of light through (transmits)
the slide.
✔ As the light passes through it is affected by the
structures and objects on the slide.
✔ These effects result in only certain parts of the light
beam being transmitted through certain parts of the
slide.
✔ This transmitted beam is then projected onto the
viewing screen, forming an enlarged image of the slide.

101
TEMs work the same way except that they shine a
beam of electrons (like the light) through the
specimen(like the slide).
Whatever part is transmitted is projected onto a
phosphor screen for the user to see.

A more technical explanation of a typical TEMs

workings is as follows (refer to the diagram):

102
103
1. The "Virtual Source" at the top represents the electron
gun, producing a stream of monochromatic electrons.
2. This stream is focused to a small, thin, coherent beam by
the use of condenser lenses 1 and 2.
✔ The first lens(usually controlled by the "spot size knob")
largely determines the "spot size"; the general size range
of the final spot that strikes the sample.
✔ The second lens(usually controlled by the "intensity or
brightness knob" actually changes the size of the spot on
the sample; changing it from a wide dispersed spot to a
pinpoint beam.

104
3. The beam is restricted by the condenser aperture
(usually user selectable), knocking out high angle
electrons (those far from the optic axis, the dotted
line down the center).
4. The beam strikes the specimen and parts of it are
transmitted.
5. This transmitted portion is focused by the objective
lens into an image.
6. Optional Objective and Selected Area metal apertures
can restrict the beam;
✔ Objective aperture enhances contrast by blocking out
high-angle diffracted electrons
✔ Selected Area aperture enables the user to examine
the periodic diffraction of electrons by ordered
arrangements of atoms in the sample.

105
7. The image is passed down the column through the
intermediate and projector lenses, being enlarged all the
way.
8. The image strikes the phosphor image screen and light
is generated, allowing the user to see the image.
✔ The darker areas of the image represent those areas of
the sample that fewer electrons were transmitted
through (they are thicker or denser).
✔ The lighter areas of the image represent those areas of
the sample that more electrons were transmitted
through (they are thinner or less dense)

106