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Dr ATIKAH, MSiJURUSAN KIMIA FMIPA-UB
2011
BIOSENSOR
A SENSOR may be defined as a device
capable of continously and reversibly recording a physical parameter or the concentration (activity) of a chemical or biochemical species
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.
DEFINITION
Normally a sensor devise consists of 3
components A recognation elemen A transduction element A processing unit
Analyte ofinterest Interfering species
Biocomponent
Transducer
ProcessorSignal
A transduction element
A part of the sensor that can transform the recognation process into a measurable signal (usually electrical or optical exp: Electrochemistry; potentiometry; spectrophotometry
A processing unit
A unit that can amplify of the primary signal converts it into a unit familiar to the analyst exp: pH, concentration (ppm, M)
a recent IUPAC definition of Biosensor:
“A self-contained integrated device which [sic] is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element which is in direct spatial contact with atransducer element
So what is an biosensor?
BIOCOMPONENTS
Enzymes Antibodies Membranes Organelles Cells Tissues Cofactors
TRANSDUCERS Electrochemical Optical Piezo-electric Calorimetric Acoustic
BIOSENSOR TYPES
Enzyme/metabolic biosensors Enzyme and cell electrodes
Bioaffinity sensors Antibodies Nucleic acids Lectin
Enzyme/Metabolic Sensors
Enzymes are biological catalysts. There are five main classes of enzymes. Oxidoreductases Transferases Hydrolases Lyases Isomerases
Oxidoreductases
Dehydrogenases Oxidases Peroxidases Oxygenases
Enzyme/Metabolic Sensors
Substrate + Enzyme
Substrate-enzyme complex
Product + Enzyme
Substrate consumption/product liberation is measured and converted into quantifiable signal.
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.
AntibodyAnalyte of interest(antigen)
Interfering species
Antibody-antigen complex
Properties of biosensors
1) The biological component must be specific and stable.
2) The reaction should be as independent of physical parameters such as pH, temperature and stirring as possible.
3) The response should be accurate, precise and reproducible.
4) The sensing element should be tiny and biocompatible.
5) The complete unit should be cheap and portable.
Design Features of Biosensors Biosensors usually have the following features:
a) Biocatalyst - converts the analyte into product. b) Transducer - detects the occurrence of the reaction and converts it into an electrical signal.c) Amplifier - amplifies the usually tiny signal to a useable level.d) Microprocessor - signal is digitised and stored for further processing, e.g. integration, derivatisation, etc.e) Display - usually need a real-time display of the analyte concentration
The Biological Component
The biological component of a biosensor can be : whole microbial cells, tissue slices, antibodies or enzymes, biosensors have been successfully constructed
using all of these materials.
1) Whole microbial cells
These are often used when the desired enzyme activity is unstable or difficult to purify.
Use of whole microbial cells results in increased stability but decreased selectivity.
This can be a either a disadvantage or an advantage, for example, in environmental monitoring a range of analytes might be detected.
1) Whole microbial cells
Biosensors based upon whole micro-organisms frequently have slow response times and they need frequent recalibration.
Usual practice is to preincubate the sensor with the analyte of interest allowing induction of the necessary enzyme systems.
1) Whole microbial cells Whole cell biosensors have been
constructed to analyse: alcohols, ammonia, antibiotics, biological oxygen demand (BOD), enzyme activities, mutagenicity, nitrates, organic acids, peptides, phosphate, sugars and vitamins.
2) Tissue slices
Sections of mammalian or plant tissue can also be used in biosensors.
This usually results in a biosensor with greater selectivity than with bacterial cells as plant and animal cells are not as metabolically versatile.
Tissue slices used to date include the
following:
3) Antibodies
Immunosensors are based on ELISA technology.
They can be very sensitive with detection levels as low as 10-21 moles in certain cases.
Immunosensors also display a very high degree of selectivity.
It is possible to use monoclonal antibodies against virtually any desired analyte.
4) Enzymes
Enzymes are the most widely used biological component and a wide range of enzymes have been successfully used in biosensors.
The advantages of enzymes are principally a combination of selectivity and sensitivity.
They also allow a wide range of transduction technologies to be used.
Examples of enzymes used to date include:
Examples of enzymes used to date include:
Transducers
Electrochemical Potentiometric Amperometric Conductimetric
1) Potentiometric Biosensors
These are usually based on ion-selective electrodes.
Such devices measure the release or consumption of ions during a reaction.
The simplest potentiometric biosensor is based on a pH-probe:
Enzyme based sensor
A potentiometric urea sensor may consist of two pH sensors one with the enzyme coated on aits surface and one without (the reference electrode)
The electrode with the urease will sense a local pH change
The pH difference bewteen the two electrodes is proportional to the urea concentration
As an example two IrOx electrodes may be used
V
urease
IrOxIrOx
Glucose sensor
Reaction
Enzyme based sensor
Enzymes are high-molecular weight biocatalysts (proteins) that increase the rate of numerous reactions critical to life itself
Enzyme electrodes are devices in which the analyte is either a substrate (also called reactant) or a product of the enzyme reaction, detected potentiometrically or amperometrically
Example : glucose sensor substrate (glucose) diffuses through a membrane to the enzyme layer where glucose is converted
Both oxygen (which is being consumed) and H2O2 (which is being produced) can be measured electrochemically (in an amperometric technique), or the local pH change can be monitored (in a potentiometric measurement)
Glucose H2O2 + gluconic acid
Glucose oxidase (in presence of oxygen)
Pt- anode (+)
Ag cathode (-)
Immobilized glucose oxidase (e.g. in cellulose-diacetate with heparin)
Polyurethane membrane
Enzyme based sensor
Amperometric glucose sensor based on peroxide oxidation,
Plateau of limiting current is proportional to the peroxide concentration which in turn is proportional to glucose - - - typical 0.6 to 0.8 V vs Ag cathode
Glucose oxidase is an oxidase type enzyme, urease is a hydrolytic type enzyme:
-
i
l
Anodic
Cathodic
+i
-i
+
+ 0.6 V
Urease
CO (NH2 )2 CO2 + 2 NH3
H2O
Reaction:
9 9 9
a) semipermeable membrane; b) entrapped biocatalyst; c) glass membrane of a pH-probe; d) pH-probe; e) electrical potential; f) Ag/AgCl electrode; g) dilute HCl; h) reference electrode
Several enzymatic reactions can be monitored by ion-selective
electrodes:
1) Detection of H+ cation
2) Detection of NH4
+ cation
3) Detection of CN- anion
The response of such electrodes is given by Nerst equation :
Where: E = measured potential (volts); E0 = characteristic constant for the electrode;R = gas constant; T = temperature (K); z = ionic charge; F = Faraday constant; [i] = concentration of uncomplexed ionic species
An increase of 59mV is seen for every order of
magnitude increase in H+ at 25o C. The logarithmic nature of the response means
that such electrodes give a wide range of detection at low accuracy and precision, usually in the range of 10-4 to 10-2 M.
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.
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.
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.
Ag/AgCl reference electrode
Internal aqueousfilling solution
Membrane/salt bridge
Porous membrane containing ionophore
Liquid ion exchanger
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.
POTENTIOMETRIC BIOSENSORS
Urea + H2O + H+ urease
2NH4+ + HCO3
-
Creatinine + H2O creatininase
N-methylhydantoin + NH4+
Penicillinpenicillinase
Penicillonic Acid
In contact with pH electrode.
Immunosensors
Affinity pairs: An enzyme/ substrate combination is only one example of an affinity pair, in nature there are many other examples of affinity pairs based on molecular recognition (think about double stranded DNA)
Affinity pairs exhibit tremendous binding selectivity for each other through their intricate 3D molecular structures (lock and key)
A much more selective affinity pair than enzyme / substrate pair is the antigen/antibody pair (AgAb) -- KA (affinity constant) values of 106-1012 LM-1 vs 102-106 LM-1 (as a consequence enzyme sensors may be reversible while imunosensors are irreversible but much more selective)
In an immunosensor one measures the concentration of either an antibody or an antigen by measuring an event triggered by the binding of an antigen/antibody- usually a label is involved (e.g. an enzyme, an isotope, a chromophore, etc.) , a direct detection of the binding event (without label) is very difficult but is being attempted in various research labs.
Immunosensors
One example of an immunosensor is an enzyme based immunosensor where the label is an enzyme--see next slide
Typically an antigen (the same antigen we are trying to determine in the unknown solution) is labeled with an enzyme (say catalase) and added to the unknow sample in which the sensor is placed
The labeled antigen competes with native (unlabeled antigen) for reaction with the antibody, which is immobilized on an electrode surface
Unbound labeled antigen is washed off and substrate for the enzyme (H2O2 in the case of catalase) is added
The enzyme decomposes H2O2 and the oxygen is picked up by the underlying oxygen sensor
The oxygen current decreases with increasing concentration of the nonlabeled native antigen in the sample solution
The enzyme reaction will produce many detectable species per bound AbAg pair, hence the name “enzyme amplification.”
Oxygen sensor
Oxygen permeable membrane
Immobilized antibody
Competition for sites on the antibody
Immunosensors
Oxygen sensor
Oxygen permeable membrane
Immobilized antibody
Antigen
Enzyme labeled antigen
Immunosensors
Oxygen sensor
Oxygen permeable membrane
Wash the unbound antigen away and add H2O2
The oygen signal is lower the higher the amount of native antigen
Oygen is formed
2-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
e flow
Working Electrode
Auxiliary Electrode
Reference Electrode(e.g. Ag/AgCl, SCE)
(e.g. Pt wire)
(e.g. Pt, Au, C)
Stirbar
Buffer solution (e.g. Tris, DPBS, Citrate)incorporating electrolyte(e.g. KCl, NaCl)
Example
Glucose + O2GlucoseOxidase
Gluconic Acid + H2O2
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.
0
50
100
150
5 10 15 20
I (nA)
[Glucose], mM
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.
Clark Oxygen Electrode-+
Platinum cathode
Polyethylene membrane
Silver anode
Electrode body
KCl soln.
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.
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
AMPEROMETRIC BIOSENSORS1. 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.
AMPEROMETRIC BIOSENSORS
2. MediatorsMany oxidase enzymes can utilise artificial electron acceptor molecules, called mediators.
A mediator is a low molecular weight redox couplewhich 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 andhence properties, including a range of redox potentials.
CV of ferricyanide 10mM
-0,00003
-0,00002
-0,00001
0
0,00001
0,00002
0,00003
-1 -0,5 0 0,5 1
mV applied
Am
ps
det
ecte
d
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)
AMPEROMETRIC BIOSENSORS
3. Metallised electrodes
The purpose of using metallised electrodes is to createconditions in which the oxidation of enzymatically generated H2O2 can be achieved at a lower appliedpotential, by creating a highly catalytic surface.
In addition to reducing the effect of interferents, dueto the lower applied potential, the signal-to-noise ratio is increased due to an increased electrochemicallyactive area.
AMPEROMETRIC BIOSENSORS
Metallisation is achieved by electrodepositing the relevant noble metal onto a glassy carbon electrodeusing cyclic voltammetry.
Successful results have been obtained from a few noble metals - platinum, palladium, rhodium and ruthenium being the most promising.
AMPEROMETRIC BIOSENSORS
Res
pons
e
Potential Potential
Res
pons
e
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.
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.
AMPEROMETRIC BIOSENSORS
Examples of commonly used polymers
are:
polypyrrole polythiophene polyaniline diaminobenzene polyphenol
AMPEROMETRIC BIOSENSORS
Electrochemical Transducers
3. ConductimetricConductimetric 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.
K+
K+
K+
A-
A-
Amperometric Biosensors
Amperometric biosensors work by enzymatically generating a current between two electrodes:
2) Amperometric Biosensors
a) applied potential; b) platinum cathode; c) silver anode (annular);d) saturated solution of KCl; e) biocatalyst; f) acetate membrane (permeable to oxygen only); g) analyte solution; h) polycarbonate membrane (permeable to oxygen, substrates and products); i) a current is generated between the electrodes
2) Amperometric Biosensors
The simplest design is based on the Clark oxygen electrode.
This has a platinum cathode and a silver/silver chloride anode.
Oxygen is reduced at the platinum cathode:
2) Amperometric Biosensors
Oxygen is consumed at the cathode generating a concentration gradient between the electrode and the bulk solution.
The rate of electrochemical reaction is, therefore, dependant on the [oxygen] in solution.
A typical application of this kind of biosensor is the glucosensor based on
immobilised glucose oxidase:
Glucose can be monitored either by the decrease in [O2] or by measuring the H2O2 by oxidation at the platinum electrode. In this case it is necessary to make the platinum electrode the anode by + 0.7v:
The most significant problem with amperometric biosensors is the dependence on dissolved oxygen, although this can be overcome by the use of mediators.
Mediators are electron transfer molecules that shuttle electrons from the enzyme to the electrode:
Some suitable mediators are:
a) ferroceneb) N-methylphenazinium cation (NMP+)c) tetracyanoquinodimethane radical anion (TCNQ.-)
Amperometric Biosensors
The electrode can be coated with NMP+TCNQ.-.
This forms an electrically conducting organic salt.
This salt binds to flavoenzymes giving efficient conduction of electrons to the electrode.
It is also possible to covalently attach the mediator to the enzyme.
This elegant approach has been done with ferrocene attached to glucose oxidase and D-amino acid oxidase:
From ISFET to ISN’T FET
Homework
1. Design a combination glass electrode. Explain how it works.
2. Design a planar immunosensor. How could you incorporate a good reference?
3. Explain how a potentiometric CO2 sensor works.
4. List a list of reasons why the ISFET did not become a commercial success.
Overview of biosensor
technology Classes of biosensor devices External analysis/detection o Large instruments o Objectives
Maximum sensitivity Highest throughput
o Samples probed Biochemical Cell populations Intracellular (single cells)