1. PhD Course Biosensors – fundamentals & applications
TOPICS IN (NANO) BIOTECHNOLOGY
Biosensors – fundamentals & applications
June 30th 2003
PhD Course

2. 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.

3. Signal Analyte of interest Interfering species Biocomponent Transducer
Processor

4. So what is an biosensor?

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

6. BIOCOMPONENTS Enzymes Antibodies Membranes Organelles Cells Tissues
Cofactors

7. TRANSDUCERS Electrochemical Optical Piezo-electric Calorimetric
Acoustic

8. BIOSENSOR TYPES Enzyme/metabolic biosensors Bioaffinity sensors
Enzyme and cell electrodes
Bioaffinity sensors
Antibodies
Nucleic acids
Lectin

9. Enzyme/Metabolic Sensors
Enzymes are biological catalysts. There are five main classes of enzymes.
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases

10. Oxidoreductases
Dehydrogenases
Oxidases
Peroxidases
Oxygenases

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

12. 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.

13. Analyte of interest
(antigen)
Antibody
Interfering species
Antibody-antigen complex

14. Transducers
Electrochemical
Potentiometric
Amperometric
Conductimetric

15. 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.

16. POTENTIOMETRIC BIOSENSORS
E = Eo + RT/nF ln[analyte]
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
ln[analyte] is the natural logarithm of the analyte activity.

17. 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.

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

19. 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.

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

21. 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

22. incorporating electrolyte (e.g. KCl, NaCl)
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

23. Example Glucose + O2 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.

24. I (nA)
150
100 50 5 10 15 20
[Glucose], mM

25. 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.

26. Clark Oxygen Electrode- +
Electrode body
Silver anode
KCl soln.
Polyethylene membrane
Platinum cathode

27. 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.

28. 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

29. 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.

30. 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.

31. AMPEROMETRIC BIOSENSORS

32. 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)

33. 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.

34. 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.

35. Glassy carbon electrode Metallised GCE
Response
Potential
Potential
Response
Metallised GCE
Glassy carbon electrodes do not catalyse the oxidation of
hydrogen peroxide.
GCEs metallised with ruthenium, rhodium, palladium or
platinum do.

36. 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.

37. AMPEROMETRIC BIOSENSORS
Examples of commonly used polymers are:
polypyrrole
polythiophene
polyaniline
diaminobenzene
polyphenol

38. 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.

39. K+ K+ K+ A- A-

40. 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...

41. 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

42. OPTICAL BIOSENSORS Disadvantages include:
ambient light is a strong interferent
fibres are very expensive
indicator phases may be washed out with time

43. 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 Snell’s Law.

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

45. 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.

46. OPTICAL BIOSENSORS
Jacket
qmax
Cladding
Core
Cladding
Jacket

47. 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.

48. 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.

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

50. 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.

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

52. 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.

53. OPTICAL BIOSENSORS Fluorescent-labelled recognition molecule
Free antigen
Immobilised analyte

54. 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).

55. 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.

56. Quartz wafer
Gold
Electrical contacts

57. 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.

58. 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.

59. 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

60. Enzyme Substrate -DH (kJ/mol)
Catalase Hydrogen peroxide
Cholesterol oxidase Cholesterol
Glucose oxidase Glucose
Hexokinase Glucose
Lactic dehydrogenase Pyruvate
b - Lactamase Penicillin G
Urease Urea
Uricase Uric acid

61. polyurethane insulation Aluminium block
Recorder
Bridge/Amplifier
Sample
Thermistor
Buffer
stream
Enzyme reactor
Heat exchanger
polyurethane insulation
Aluminium block