created with mindgenius business 2005® biosensors dr. alison willows

Created with MindGenius Business 2005®

Biosensors

Dr. Alison Willows


BiosensorsIUPAC definition:

A device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals.


Recommended reading

Eggins (2002) Chemical Sensors & Biosensors. Wiley

Cooper & Cass (2004) Biosensors, 2nd edn. Oxford University Press

Wang (2006) Analytical Electrochemistry, 3rd edn. John Wiley & sons.


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Introduction to electrochemical measurements

Electroanalysis – the application of electrochemistry to solve real-life analytical problems

There are essentially three main types of measurement: Conductimetric – measures solution resistance to

obtain the concentration of charge. It is not species selective and has limited uses.

Potentiometric - measures the equilibrium potential of an indicator electrode against a selected reference electrode using a high impedance voltmeter.

Amperometric/voltammetric – measures current at either a fixed potential (amperometric) or over a range of potentials (voltammetric)


Introduction – Electrochemical Cell

All electrochemical cells contain at least two electrodes, many contain three.

For a 2 electrode system a combined secondary and reference electrode is used


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Reference Electrodes

Is used to give an arbitrary zero, to determine a potential difference.

Potential must be stable over time and not change with temperature

Standard Hydrogen Electrode defines the standard electrode potential scale against which all other reference electrodes are measured


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Reference elecrtodesStandard Hydrogen Electrode


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Reference ElectrodesStandard Hydrogen Electrode (cont.) Very reproducible Consists of Pt foil that is platinised

(platinum black deposit) H+ + e- ½H2 Set-up is made more difficult by need

for constant stream of hydrogen gas Other reference electrodes are more

commonly used


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Reference ElectrodesSaturated Calomel Electrode (SCE) Reference potential depends on the concentration

of chloride within the KCl solution so a saturated solution is usually used

Reference potential at 25°C should be +0.242V versus SHE


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Reference ElectrodesSilver/silver chloride electrode (Ag/AgCl) Very widely used due to its simplicity A silver surface is anodised in a

saturated solution of KCl, this creates a silver chloride layer due to oxidation

Ag+(aq) + Cl-(aq) AgCl(s)


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Mass Transport and Electron Transfer

Movement of a reactant from bulk solution to the electrode surface and to bulk solution again is governed by electron transfer and mass transport.

There are three forms of mass transport which can influence a reaction:

Diffusion – occurs due to concentration gradients

Convection – results from the action of a force on the solution. Natural or forced

Migration – an electrostatic effect due to the application of a voltage on the electrodes


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Diffusion Occurs in all solutions due to local

uneven concentrations of reactants As the conversion reaction occurs

only at the electrode surface there will be a lower reactant concentration at the electrode than in bulk solution

Similarly, a higher concentration of product exists near the electrode than further out into the solution


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Convection This results from the action of a force on the

solution. There are two forms of convection, natural and forced.

Natural Convection – present in any solution, generated by small thermal or density differences. Acts to mix the solution in a random and therefore unpredictable manner

Forced Convection – typically several orders of magnitude greater than any natural convection effects.

Forced convection removes the random aspect if convection is introduced in a well-defined and quantitative manner so that it can be mathematically modelled and is reproducible


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Convection

Examples of forced convection techniques

Rotating Disc Electrode Wall Jet Electrode

Electrode is rotated to produce reproducible flow of solution to the electrode surface

Solution is forced onto the electrode surface


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Migration

An electrostatic effect arising due to the application of a voltage on the electrodes creating a charged interface (the electrodes)

Any charged species near that interface will either be attracted or repelled from it by electrostatic forces

Migration is notoriously difficult to calculate for real solutions

Most voltammetric measurements are performed in solutions containing a background electrolyte

(This is a salt (eg KCl) which does not undergo electrolysis itself)

Adding a large quantity of the electrolyte (relative to the reactants) ensures migration will not significantly affect the electrolysis reaction


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Mass Transport To model quantitatively the current flowing at the

electrode we must account for the electrode kinetics, the 3 dimensional diffusion, convection and migration of all species involved

This is currently beyond the capacity of even the fastest computers - and will be for some time

Careful design and control of the electrochemical experiment can help to simplify the mass transport effects Migration is effectively stopped by addition of

background electrolyte Use of hydrodynamic electrodes provides well-defined

convective regime Use of microelectrodes or short experiment time


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Interfacial region - double layer

When a potential is applied and electrons are pumped into or out of an electrode the surface becomes charged

This charged surface attracts ions of opposite charge

The charged electrode and the oppositely charged ions next to it are known as the electric double layer

Example - negatively charged electrode


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BiosensorsReference: Eggins (2002) Chemical Sensors & Biosensors.

Wiley Uses a biological recognition element

which may also catalyse the electrode reaction

(e.g. enzyme, antibody, lectin, whole cells)

For many analytes the electrode reaction is too slow to be useful for analysis

E.g. glucose is only slowly oxidised on most electrode materials


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Biosensors

There are three modes of oxidation reactions that occur in biosensors

First generation: oxygen electrode based

Second generation: mediator based Third generation: directly coupled

enzyme


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Biosensors – first generation Original glucose enzyme electrode used

molecular oxygen as the oxidizing agent

Glucose is oxidised to gluconic acid (which forms gluconolactone) by molecular oxygen, catalysed by glucose oxidase, GOD, from Aspargillus nigus.

GOD is a flavin enzyme therefore the redox centre is Flavin Adenine Dinucleotide (FAD) represented by:

2 2 2glucose gluconic acidGODO H O+ ¾¾¾® +

22 2oxidised reduced

FAD e H FADH- ++ + ƒ

GOD


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Biosensors – first generation

H2O2 is electrochemically active and can be detected on Pt electrodes either by oxidation (typically +600 mV vs Ag/AgCl) or by reduction

First generation biosensors used oxidase enzymes and measured the resulting H2O2 usually by oxidation

reduce reduce oxidise oxidise

H2O2

O2

GOD/FAD

GOD/FADH2

Glucose

Gluconolactone


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red red ox ox

H2O2

O2

GOD/FAD

GOD/FADH2

Glucose

Gluconolactone

Semi-permeable membrane

ox

Electrode surface

2e-

First generation all involve five steps in the reaction:

i. Diffusion from solutionii. Membrane transportiii. Reaction of enzyme with substrateiv. Reaction of enzyme with mediator (O2(aq) in this case)

v. Reaction of mediator with the electrode


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Mass transport in solution or in the membrane should be the rate determining step for reliable concentration sensors

Therefore the design criteria requires: High enzyme concentration in the

sensor Very high enzyme activity


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Ideally, linear range should coincide with expected analyte concentrations

Current is rate i/nFA is flux in Fick’s Law

cu

rren

t

[substrate]

Enzyme is saturated

Linear region

Can extend linear range by using more enzyme, more active enzyme or decreasing membrane permeability


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Deficiencies: Dependance on [O2(aq)] at low [O2(aq)]

causes problems in anoxic or ischaemic environments

+600 mV (vs Ag/AgCl) does not allow good sensitivity. E.g. ascorbic acid is oxidised on most electrode materials at potentials <600 mV and is present in most enzyme or cell preparations


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Biosensors – second generation Other mediator species were investigated:

Ferrocene mediated systems Quinone/dihydroquinone mediated systems

Generally,

Where M is a mediator

red ox

MR

MO

enzyme/FAD

enzyme/FADH2

substrate

product ne-


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Biosensors – second generation

A good mediator should React rapidly with the enzyme Show reversible (i.e. fast) electron transfer

kinetics Have a low over-potential for re-generation Be independent of pH Be stable in both its oxidised and reduced

forms Not react with oxygen Be non-toxic

Ferrocenes meet all these criteria and are easily modified by aromatic substitution on the cyclopentodienyl rings.

Such modifications affect solubility and redox potential

Example: ExacTech (medisense) glucose sensor


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Biosensors - third generation

Why is a mediator needed to couple an enzyme to an electrode?

Is it possible to reduce/oxidise an enzyme directly at an electrode?

The redox centre of the protein is too far from the electrode surface to enable significant rates of electron transfer, i.e. a high overpotential is required

Adsorption – proteins are generally surface active and are electronic insulators


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Biosensors – third generation

Adsorption causes de-naturation of protein (change in tertiary structure, shape) Loss of active site, shape/orientation FAD centre either falls off or is moved too far

from the active site

Active site

2/3 hrs 2-12 hrs


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Biosensors – third generation

Some possible solutions Modify the electrode surface

Thin electronically conducting polymers can be deposited on the electrode surface (e.g. polypyrrole, polyaniline)This is used to immobilise the enzymeThe polymer prevents protein deposition on the electrode

Organic-conducting-salts E.g. tetrathiafulvalene (TTF) is reversibly oxidised while

tetracyanoquinodimethane (TCNQ) is reversibly reduced. A pair of these molecules forms a charge-transfer

complex When incorporated into an electrode the surface

becomes highly reversible and stable to many enzymes


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Biosensors – inhibition based sensors“Chemical canaries”

Will only give a qualitative YES/NO response

“concentration devices” have LoDs ~ >10-100 mol dm-3

Rate determining step is substrate diffusion

Enzyme (ox)

Enzyme (red)

substrate

product

to electrode


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Biosensors – inhibition based sensors

“Chemical canaries” Enzyme turnover numbers are typically in

the range 103 – 105 s-1. One inhibitor molecule can kill/block etc

one enzyme molecule If enzyme is inhibited by analyte,

substrate concentration will rise rapidly Amplification will be of a similar order to

the turnover number For inhibition sensors, the enzyme and

substrate reaction is rate determining step


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“Chemical canaries”Example: CN- sensitive chemical canary

Current used to regenerate Fc+ Fc is recorded

H2O2

O2 H2O

Fc+

Fc 2e-

HRP

-0.6 V 0 V

e-


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“Chemical canaries” CN- inhibits HRP and leads to a fall in

catalytic current

There is no simple relationship between measured current and [CN-] because the enzyme-substrate reaction is the rate determining step

CN-

t

-i


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“Chemical canaries”Example: Hydrogen Sulfide, H2S

Cytc(III)

Cytc(II)

Cyt OD (red)

Cyt OD (ox)

O2

H2O2

e-

Aldrithiol4 monolayer (enables direct e- transfer)

Au electrode


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“Chemical canaries” Cyt OD is inhibited by H2S

Reversible inhibition so H2S can be washed off Similar devices exist for organophosphates (nerve

gases, insecticides) using acetylcholinesterase and chemical reactions coupled to this

H2S

t

i

buffer

created with mindgenius business 2005® biosensors dr. alison willows

Documents

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used slide

selected reference electrode

electrode surface

indicator electrode

electrode system

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bulk solution