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PhD Course Biosensors – fundamentals & applications

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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

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

7 TRANSDUCERS Electrochemical Optical Piezo-electric Calorimetric

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

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.

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.

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

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.

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

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

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

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.

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

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.

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.


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

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.

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.

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.

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

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

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