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TOPICS IN (NANO) BIOTECHNOLOGY Biosensors – fundamentals & applications


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

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TOPICS IN (NANO) BIOTECHNOLOGY Biosensors – fundamentals & applications

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PhD Course

TOPICS IN (NANO) BIOTECHNOLOGY

Biosensors – fundamentals & applications

June 30th 2003


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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.


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Analyte of

interest

Interfering species

Biocomponent

Transducer

Signal

Processor


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So what is an biosensor?


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HISTORY

  • 1962, Clark and Lyons

  • 1967, Updike and Hicks

  • 1969, Guilbault

  • 1972, Reitnauer

  • 1975, Yellow Springs Instrument

  • 1975, Janata

  • 1979, Danielsson


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BIOCOMPONENTS

  • Enzymes

  • Antibodies

  • Membranes

  • Organelles

  • Cells

  • Tissues

  • Cofactors


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TRANSDUCERS

  • Electrochemical

  • Optical

  • Piezo-electric

  • Calorimetric

  • Acoustic


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BIOSENSOR TYPES

  • Enzyme/metabolic biosensors

    • Enzyme and cell electrodes

  • Bioaffinity sensors

    • Antibodies

    • Nucleic acids

    • Lectin


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Enzyme/Metabolic Sensors

  • Enzymes are biological catalysts. There are five main classes of enzymes.

  • Oxidoreductases

  • Transferases

  • Hydrolases

  • Lyases

  • Isomerases


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Oxidoreductases

  • Dehydrogenases

  • Oxidases

  • Peroxidases

  • Oxygenases


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Enzyme/Metabolic Sensors

  • Substrate + Enzyme

Substrate-enzyme complex

  • Product + Enzyme

Substrate consumption/product liberation is measured and converted into quantifiable signal.


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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.


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Analyte of interest

(antigen)

Antibody

Interfering species

Antibody-antigen complex


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Transducers

  • Electrochemical

    • Potentiometric

    • Amperometric

    • Conductimetric


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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.


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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.


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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.


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Ag/AgCl reference electrode

Internal aqueous

filling solution

Liquid ion exchanger

Membrane/salt bridge

Porous membrane containing ionophore


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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.


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POTENTIOMETRIC BIOSENSORS

urease

Urea + H2O + H+

2NH4+ + HCO3-

creatininase

Creatinine + H2O

N-methylhydantoin + NH4+

penicillinase

Penicillin

Penicillonic Acid

In contact with pH electrode.


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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


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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


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Example

Glucose + O2

Glucose

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.


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I (nA)

150

100

50

0

5

10

15

20

[Glucose], mM


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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.


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Clark Oxygen Electrode

-

+

Electrode body

Silver anode

KCl soln.

Polyethylene membrane

Platinum cathode


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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.


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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


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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.


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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.


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AMPEROMETRIC BIOSENSORS


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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)


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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.


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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.


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Response

Response

Potential

Potential

Glassy carbon electrode

Metallised GCE

Glassy carbon electrodes do not catalyse the oxidation of

hydrogen peroxide.

GCEs metallised with ruthenium, rhodium, palladium or

platinum do.


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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.


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AMPEROMETRIC BIOSENSORS

  • Examples of commonly used polymers are:

    • polypyrrole

    • polythiophene

    • polyaniline

    • diaminobenzene

    • polyphenol


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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.


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K+

K+

K+

A-

A-


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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...


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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


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OPTICAL BIOSENSORS

  • Disadvantages include:

    • ambient light is a strong interferent

    • fibres are very expensive

    • indicator phases may be washed out with time


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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.


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OPTICAL BIOSENSORS

A

Cladding

Core

B

B

Total Internal Reflection


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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.


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OPTICAL BIOSENSORS

Jacket

qmax

Cladding

Core

Cladding

Jacket


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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.


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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.


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OPTICAL BIOSENSORS

  • There are basically two different configurations used at the tip of the fibre-optic probe

    • the distal cuvette configuration

    • waveguide binding configuration


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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.


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OPTICAL BIOSENSORS

Fibre-optic probe

Distal cuvette

Detection molecule

Analyte


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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.


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OPTICAL BIOSENSORS

Fluorescent-labelled

recognition molecule

Free antigen

Immobilised analyte


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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).


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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.


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Quartz wafer

Gold

Electrical contacts


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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.


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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.


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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


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EnzymeSubstrate-DH (kJ/mol)

CatalaseHydrogen peroxide100.4

Cholesterol oxidaseCholesterol52.9

Glucose oxidaseGlucose80.0

HexokinaseGlucose27.6

Lactic dehydrogenasePyruvate62.1

b - LactamasePenicillin G67.0

UreaseUrea6.6

UricaseUric acid49.1


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Recorder

Bridge/Amplifier

Sample

Thermistor

Buffer

stream

Enzyme reactor

Heat exchanger

polyurethane insulation

Aluminium block