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Biosensors

Biosensor : Any device that uses specific biochemical reactions to detect

chemical compounds in biological samples.

A sensor that integrates a biological element with a physicochemical

transducer to produce an electronic signal proportional to a single analyte
which is then conveyed to a detector.

Based on specificity and sensitivity of biological systems

a) Thin layer of active biological material in contact with electrical transducer

b) Transducer converts observed change (physical / chemical) in quantifiable signal

c +d+ e )Electronic signal magnitude proportional to concentration of a specific
compound

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Biocatalysis (Enzyme/metabolic): Substrate consumption/product liberation is measured

and converted into quantifiable signal.

Bioaffinity: 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.

Biosensors applications
1. Control of critical metabolites during surgery
2. Ambulatory and Hospital Emergencies:
– Obviously expensive and time consuming analysis in central
laboratories
– Accelerates the diagnosis and early treatment
– Less risk of damage to the sample
3. Domestic Diagnosis:
• Pregnancy test
• Glucose Control in Diabetics

4. In vivo applications :
– artificial pancreas
– Correction metabolite levels
– Problems: Miniaturization and Biocompatibility
5. Industrial, military ans environmental applications:
– Food industry
– Cosmetics
– Fermentations control
– Quality control
– Explosives Detection
– Detection of nerve gases and / or biological toxins
– Pollution control
–

– .

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TYPES OF BIOSENSORS
1. Electrochemistry Biosensors
– Amperometric: measurement of electrical currents associated
with electrons involved in redox processes
– Potenciometric: ion-selective electrodes
– Conductimetric: measurement of conductivity changes
associated with changes in the ionic environment of the
solutions
2. Termometric Biosensors
3. Piezoelectric biosensors
4. Optical biosensors
Evanescente wave
Surface plasmon resonance

5. Cell Biosensors
6. Immunosensors

An enzyme electrode
P1 P1 -

S1 -

Sample E Electrode
S2 -

P2 P2 -
I P2*

Protective Enzyme layer Permselective

membrane membrane

1. Thin enzyme layer with high specific activity,

2. Good selection of membranes
Response controlled by diffusion through the permselective membrane
(not by enzyme kinetics)
Enzyme activity low - thick membrane needed to achieve linear response, response slow
Enzyme activity high - thin membrane OK, rapid response

3. Detection: Steady-state or flow injection analysis

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Biosensor parameters
1. Sensitivity
dS/dt 2. Linear response
3. Detection limit
Analyte Signal
4. Background noise
5. Baseline drift
DS 6. Selectivity
7. Response
8. Operating stability
Time Background noise 9. Shelf life

Detection Analyte
S limit
N
DS/Dc Linear response S=3N

Assay of the detection limit

c

Response time and kinetics in biosensors

RESPONSE TIME : Time necessary for having 95% of

the response.
Recovery Time: Time necessary to recovery the
baseline.

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Screen printing

Mobile wiper

Matrix carrier
Paste

Screen grid

The sensor body is made from ceramics.

A gold working electrode (a)
is surrounded by an Ag/AgCl reference
Screen print electrode (b)
and gold auxiliary electrode (c).
https://www.youtube.com/watch?v= Silver output contacts (d).
GxThyTTztmQ The ruler in the bottom is in millimeter
scale

Electrode designs
A. Surface modified electrodes
Electrode material: graphite, glassy carbon, gold, SnO2
Coupling: carbodiimide, glutaraldehyde, adsorption

B. Polymer-based electrodes
Crosslinking with Os(bpy)23+/2+ redox polymer,
electropolymerized polypyrrole, o-phenylethylamine

C. Bulk modified composite electrodes

Graphite-silicone oil paste, paraffin oil paste,
epoxy composite

D. Tissue-modified carbon paste electrodes

Asparagus tissue, tobacco callus tissue,
horseradish root, kohlrabi skin

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1. Electrochemistry Biosensores:
1.1 Amperometric
• Enzyme catalyzed redox reactions
• Constant voltage between two electrodes
• Current due to the reaction on the electrodes

O2 + 2 H2 O + 2 e- H2 O2 + 2 OH- A) Hard epoxy resin

2 H2 O2 + 2 e- 2 OH- (B) platinum cathode in the center of a projection.
(C) silver anode in a circular
(D) Rubber ring spacer holding a paper soaked in an
Cat o d e ( Pt ) Bridge KCl electrolyte and a polytetrafluoroethylene membrane
separating the electrodes from the reaction mixture.

Between the central platinum cathode and the

4 e- surrounding anode of silver applied potential of 0.6
A no d e ( A g ) volts. The circuit is closed with saturated KCl solution.
- +
0 ,6 - 0 ,7 v The molecular oxygen dissolved in the cathode is
reduced in the platinum catode.
4 Ag 4 A g + + 4 e-
Electrons are released and produces electrical
4 A g + + 4 Cl- 4 A g Cl current can be measured.

Glucose and sacarose determination

Glucose
Electrodo

Sacarose

O2 Fluxe
Fructose
Glucose
GOD
Glucosa-oxidasa Invertase

D-gluconolactone
response H2O2

glucose
sacarose

time

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Fish freshness:
After death, the nucleotides of fish undergo a series of progressive degradation
reactions:
ATP > ADP > AMP > IMP > HxR > Hx > Xantine > Uric Acid

The accumulation of inosine and hypoxanthine nucleotides is an

indicator of the time after the fish died and its storage conditions.
Biosensor: Xanthine oxidase and nucleoside
phosphorylase immobilized in a triacyl cellulose membrane
of an oxygen electrode

K < 20 Fresh fish can be eaten raw

20 > K < 40 Fish must be cooked
K > 40 Non-eatable fish
The nucleotides could be determined using the same sample and electrode, but adding
nucleotidase and adenosine deaminase

First generation biosensors - response to the substrates in solution

Glucose + O2 Gluconic acid + H2O2
1. Reduction of oxygen with a Clark type electrode at -0.6 V (vs SCE)
2. Oxidation of hydrogen peroxide at a Pt electrode at +0.7 V
3. Measuring of pH change
Examples of hydrogen peroxide measuring biosensor
Analyte Enzyme Reaction

Alcohol Alcohol oxidase Ethanol + O2  Acetaldehyde + H2O2

D-Glucose Glucose oxidase β-D-Glucose + O2  Gluconic acid + H2O2

Lactose Galactose oxidase Lactose + O2  Galactose dialdehyde der. + H2O2

L-Lactate L-Lactate oxidase L-Lactate + O2  Pyruvate + H2O2

Starch Amyloglucosidase Starch + H2O  β-D-Glucose

Glucose oxidase β-D-Glucose + O2  Gluconic acid + H2O2

Sucrose Invertase Sucrose + H2O  α-D-Glucose + β-D-Fructose

Mutarotase α-D-Glucose  β-D-Glucose
Glucose oxidase β-D-Glucose + O2  Gluconic acid + H2O2

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Redox mediators in amperometric

Biosensors ( 2nd generation)
• More specific oxidase reagent to
oxidize

• Fast electron transfer rates

• Ability to be regenerated easily

• Biocatalytic membrane retainable

• Not react with other molecules,

including molecular oxygen

• Tetracyanoquinodimethane: partial electron

acceptor
• Ferrocene, tetrathiofulvalene and N-methyl
phenazinium partial electron donor
• Hydroquinone and ferrocyanide soluble
mediators

Glucose biosensor

• Device sensing area with single use electrode

• Deposition on a plastic strip. Reference electrode consists of Ag / AgCl and
carbon electrode with glucose oxidase and ferrocene mediator
• Both electrodes covered with hydrophilic tissue. Passage of molecules of
different size, homogeneous diffusion, prevents uneven evaporation
• Duration 6 months
• 2-25 mM detection in blood drop
• Results in 30 seconds

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Second generation biosensors

- mediated electron transfer between enzyme and electrode
- can be easily miniaturized
Possible by the ability of pyrrole to polymerize
by electrochemical oxidation in mild conditions
enough to trap enzymes and mediators
without denaturation

Third generation biosensors

- direct electron transfer between enzyme and electrode

In the third generation biosensor the

redox cofactor of the enzyme is covalently (or
electrostatically) bound to the working
electrode. This facilitates the re-reduction
(or re-oxidation) of the enzymes after they
have interacted with their substrates and
assists in carrying electrons or holes to or from
the working electrode.

Cell-based based biosensors

- cheaper than purified enzymes,
Nocardia erythropolis cells immobilised in polyacrylamide or
agar (cholesterol oxidase)
Cholesterol + O2  Cholest-4-en-3-one + H2O2

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1.2 Potentiometric biosensors

Three types of ion-selective electrodes which are of use in

biosensors:

• Glass electrodes for cations.

• Glass pH electrodes coated with a gas-permeable
membrane selective for CO2, NH 3 or H 2S.
• The iodide electrode is useful for the
determination of I- in the peroxidase reaction.

H2O2 + 2H+ + 2I- peroxidase I2 + 2H2O

1.2 POTENTIOMETRIC BIOSENSORS

• The biosensor consists of an immobilised enzyme

membrane surrounding the probe from a pH meter.

Glucose oxidase
D-glucose + O2 D-glucono-1,5-lactone+H 2O2

H2O

D-gluconate+ H +

Penicillinase
Penicillin Penicilloic acid+H+
Urease (pH 9.5)
H2NCONH 2 + 2H 2O 2NH3 + HCO3- + H+
Lipase
Neutral lipids + H2O Glycerol + fatty acids +H +

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Potentiometric biosensors (ENFET)

enzyme-linked field effect transistors

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 insulated by a thin layer (0.1 mm thick) of silica (SiO2) which forms the gate of the FET.
Above 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.

FET
+
-
Drain Gate
Insulator Source
+ + + +
(Not conductive enough)
(Electron Channel)

- - -
- -

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FET
+
-
Threshold Voltage
Drain Gate
Insulator Source
+ + + +

FET
+

-
Drain Gate
Insulator Source
+++++ + ++
- - - - - -
- -
- - -
- -

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1.3 Conductometric Biosensors

Urea biosensor
NH2CONH2 +3H2O

Urease

2HN 4 + + HCO3- + OH-

amidases,
Immobilized urease decarboxylases,
Surgery and Renal Dialysis esterases,
Reaction causes great change in phosphatases and
ion concentration nucleases.

An alternating field between two electrodes

allows the determination of changes in
conductivity, avoiding unwanted
electrochemical processes
Electrodes arranged to occupy minimal
space
0.1 and 10 mM urea

2. Thermometric biosensors
Enzyme-catalysed reactions exhibit the same
enthalpy changes as spontaneous chemical
reactions.
Considerable heat evolution is noted (5-100kJ/mol). DT=− DHn /C
p p

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.

Reaction confined insulated devices (a) Accurate

proper insulation
The analyte stream passes through a heat exchanger
(b)
The reaction takes place in a small reactor (c)
The difference in temperature of the analyte enters
and exits the product is determined by
interconnected thermistors (d)

Differences up to 0.0001 ° C

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Reactions used in thermometric biosensors

Exemples of thermometric biosensors

Flow injection thermal biosensor array for

simultaneous determination of lactate, glucose, urea
and penicillin.

Bin Xie, Kumaran Ramanathan, Bengt Danielsson

Mini/micro thermal biosensors and other related devices for biochemical/clinical analysis and monitoring
TrAC Trends in Analytical ChemistryVolume 19, Issue 5, May 2000, Pages 340–349

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3. Piezoelectric biosensors
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).

DF = -2.3x106 F2DM/A (Sauerbrey equation)

DF = frequency change in oscillating crystal in Hz, F = frequency of piezoelectric quartz crystal
in MHz,
DM = mass of deposited film in g, and A = area of electrode surface in cm 2.

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.

Piezoelectric biosensors

Sensors configured as an oscillator can be

equipped with an antenna for remote
sensing and control.

Another advantageous feature of using

piezoelectric materials is that the same
electromechanical transduction mecha-
nism can be used not only for a sensing,
but also for actuation.
Piezoelectric biosensor: A piezoelectric transducer with biological sensing layer
(AG - AB) This property is essential for the design of
piezoelectric surgical micro-cutters or
liquid micro-flow systems.

Piezoelectric immuno-nanosensors are

inexpensive, easy-to-use, and feature rapid
response, hence they may allow for wide
screenings and the development of effective
preventive strategies for a broad range of
diseases, including bio-agent detection such as
anthrax or smallpox, viral infections, and
cancer
www.tms.org/pubs/journals/JOM/0010/Kumar/Kumar-0010.html

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Immobilization of Antibodies in a quartz crystal microbalance (QCM)

A piezoelectric immunosensor for rapid

detection of Escherichia coli O157:H7.

The immunosensor could detect the target

bacteria in a range of 103–108 CFU/ml within
30–50 min.

The cleaned crystals were immersed in

an ethanol solution of 16-
mercaptohexadecanoic acid (MHDA).
MHDA-modified crystals were treated with EDC
(Carbodiimide)-NHS (Nhydroxysuccinimide) to convert
the terminal carboxylic group to an active NHS ester.

After rinsing with water and drying,

anti-E. coli O157:H7 antibodies were added onto Au
electrodes and stored at 4 C overnight (at least 15 h).
The excess antibodies were removed by rinsing with
PBS.
X.L.Su and Y.Li, Biosensors and Bioelectronics 19 (2004) 563–574

4. OPTICAL BIOSENSORS
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

• 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

• Disadvantages include:
ambient light is a strong interference
fibres are very expensive
indicator phases may be released with time

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4.1 Fibre optics (optodes) 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 Snell’s Law.
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.

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.

Absorption at 630 nm
Optical fiber Change from yellow to greenish Optical cell
blue of bromocresol green when
bound to serum albumin at pH 3.8

Linear response to albumin in a

range of from 5 to 35 mg/cm3

Detects changes in
concentration of oxygen in
determining the reduction in
the fluorescence of a
fluorophore (quenching)

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

The waveguide binding tip configuration involves

the binding of fluorescent-labelled detector
molecules (e.g. antibodies) to covalently attached
The distal cuvette configuration involves
analyte molecules on the fibre surface.
immobilisation of detection molecules in a
As the label is close to the surface it is excited by
porous, transparent medium at the fibre tip.
the evanescent wave emanating from the fibre
and the resulting fluorescence is coupled back
The fluorescence changes when the analyte
into the fibre.
diffuses and is bound.
Free analyte competes for the binding sites on the
recognition molecules, permitting them to diffuse
Excitation comes from out of the fibre and
away from the surface with a resultant decrease in
emission is coupled back into the fibre
fluorescence.

Evanescent wave: It is based on a total internal reflection fluorescence, which consists in the
absorption and emission of photons.
In a radiation sensor that travels along a waveguide by total internal reflection creates a field
called evanescent electromagnetic field, which can penetrate a specific distance from the
surface depending on the angle of incidence at the interface and the wavelength of the
excitation radiation.

The evanescent wave in turn can interact with the medium, causing an electromagnetic field to
the medium can return with higher refractive index, resulting in changes in the light continuing
along the waveguide.
Any molecular interaction occurs in the field (such as an analyte binding to an immobilized
receptor on the surface of the waveguide) produces changes in the characteristics of the light
propagating through the waveguide that can be measured and related with the concentration
of analyte.

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Evanescent wave

(a) The evanescent wave penetrates pass the fiber core surface and excites the labeled
antibody–protein G complexes. Only the interaction between the analyte and the immobilized
molecular complexes within the evanescent wave region can be sensed.
(b) Conceptual schematic of a FRET immunosensor.
When the antibody binds with the target antigen, a conformational change in the 3D structure
of an antibody will occur, resulting in non-radiative energy transfer from the donor to
acceptor fluorophores.

4.2 Surface plasmon resonance (SPR)

Plasmons are collective oscillations of the conduction electrons in a metal.
The surface plasmon resonance occurs when a polarized light is directed from a layer of higher
refractive index (a prism) to a lower index of refraction, a metallic layer of gold or silver, which
lies between the prism and the sample. Light incident on the interface between the metal and
the prism causes a surface plasmon excitation to a given angle of incidence called resonance
angle.
The resonance angle depends strongly on the refractive index of the medium adjacent to the
metal sheet, so its variations will be proportional to the concentration.
The binding of the analyte to the recognition element is a refractive index change on the surface
of the metal and, therefore, a shift of the resonance angle. All biomolecules have refractive
properties, so no labeling required

105.16 nm

5 µm 52.58 nm

0 nm
5 µm

2.5 µm

2.5 µm

0 µm 0 µm

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Biomolecular Interaction Analysis BIACORE

The surface is divided into 4 flow cells,

which can be used individually or in a
number of combinations.

Sensor Chips Properties:

-Aqueous environment (hydrogel,
containing 97-98% water)
-Mobility (chains are not cross-linked)
-Efficient use of the evanescent field
(thickness about 100 nm)
-Increased sensitivity (more coupling
sites than on a flat surface)
-Allows covalent coupling (through
carboxyl groups)

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The ligand, is immobilised on the dextran matrix of a sensor chip. The second interactant, the
analyte, is injected in a continuous flow by means of a microfluidic system.

Molecular association and dissociation events produce variations in the SPR signal that are
recorded as resonance units (RU)as a function of time.

The mathematical evaluation of the resulting curves, called sensorgrams, allows to calculate
the kinetic parameters and the equilibrium affinity of the ligand-analyte interaction.

Change in SPR angle of 0.1= 1000 RU = 1 ng/mm2

5. Cell Biosensors
Microbial cells as biocatalysts, possess certain advantages over the purified
enzymes when used in biosensors:
cheap
Longer half-life
Less sensitive to inhibition, the pH and temperature
self-healing capacity

Disadvantages:
Slower response
Slower recovery rate
Low selectivity
easily disintegrating
Milder conditions

Very useful when you require multiple steps or the Receptor-cell-transistor

presence of coenzymes biosensor technology
Living or dead cells

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B cell lines were genetically engineered to express cytosolic aequorin, a calcium

sensitive bioluminiscent protein from jellyfish, as well as membrane-bound
antibodies specific for pathogens of interest (Rider et al., 2003)
Cross-linking of the antibodies with specific pathogens increases intracellular
calcium concentrations within seconds, causing the aequorin to emit light.
This sensor was named CANARY (cellular analysis and notification of antigen
risks and yields) and it has an excellent speed, sensitivity, and specificity.
•The CANARY Cell-based sensor
detected as few as 50 CFU of Y.
pestis in a total assay time of 3
minutes.

•The system was also tested in

one food sample-E. coli O157:H7
in lettuce.

•500 CFU/g of E. coli O157:H7 in

lettuce was detected in less than
5 minutes, including the sample
preparation.
It can distinguish pathogenic E. coli O157:H7 from
•1000 CFU of B. anthracis spores
nonpathogenic E. coli strains.

Biosensor Based on Immobilized Indicator Cells

Very fast (1-6 hours)

Good sensitivity (MOI>10:1)

Portability-collagen entrapped
cells remain viable in 48 well
plates for 48 hours.

A B-lymphocyte cell line was encapsulated in a collagen gel matrix

This assay measures alkaline phosphatase or lactate dehydrogenase released by

cells infected with pathogens or exposed to different toxins.
The system was tested using different strains of Listeria, listeriolysin O, and
enterotoxins from Bacillus species.

Banerjee et al., Laboratory Investigation (2007) 1-11.

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The main advantage of cell-based biosensors is that they provide information about
the physiological effects of pathogens/toxins.

Capable of distinguishing between viable pathogenic strains from nonpathogenic

ones or dead cells.

Sensitivity and specificity comparable to current methods.

Expected to have broad applications in food testing, animal health, biodefense,

disease diagnosis etc.

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