BIOSENSOR-2011

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.


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.

Ag/AgCl reference electrode

Internal aqueousfilling solution

Membrane/salt bridge

Porous membrane containing ionophore

Liquid ion exchanger


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.


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


Immobilized antibody

Antigen

Enzyme labeled antigen

Immunosensors

Oxygen sensor


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.


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

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.


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


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 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.


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.


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.


Examples of commonly used polymers

are:

polypyrrole polythiophene polyaniline diaminobenzene polyphenol


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


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:


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)

Documents

BIOSENSOR-2011