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Created with MindGenius Business 2005® Biosensors Dr. Alison Willows.

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Presentation on theme: "Created with MindGenius Business 2005® Biosensors Dr. Alison Willows."— Presentation transcript:

1 Created with MindGenius Business 2005® Biosensors Dr. Alison Willows

2 Created with MindGenius Business 2005® Biosensors IUPAC 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.

3 Created with MindGenius Business 2005® Recommended reading Eggins (2002) Chemical Sensors & Biosensors. Wiley Cooper & Cass (2004) Biosensors, 2 nd edn. Oxford University Press Wang (2006) Analytical Electrochemistry, 3 rd edn. John Wiley & sons.

4 Created with MindGenius Business 2005® 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)

5 Created with MindGenius Business 2005® 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

6 Created with MindGenius Business 2005® 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

7 Created with MindGenius Business 2005® Reference elecrtodes Standard Hydrogen Electrode

8 Created with MindGenius Business 2005® Reference Electrodes Standard Hydrogen Electrode (cont.) Very reproducible Consists of Pt foil that is platinised (platinum black deposit) H + + e -  ½H 2 Set-up is made more difficult by need for constant stream of hydrogen gas Other reference electrodes are more commonly used

9 Created with MindGenius Business 2005® 9 Reference Electrodes Saturated 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 V versus SHE

10 Created with MindGenius Business 2005® Reference Electrodes Silver/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)

11 Created with MindGenius Business 2005® 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

12 Created with MindGenius Business 2005® 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

13 Created with MindGenius Business 2005® 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

14 Created with MindGenius Business 2005® 14 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

15 Created with MindGenius Business 2005® 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

16 Created with MindGenius Business 2005® 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

17 Created with MindGenius Business 2005® 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

18 Created with MindGenius Business 2005® Biosensors Reference: 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

19 Created with MindGenius Business 2005® 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

20 Created with MindGenius Business 2005® 20 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: GOD

21 Created with MindGenius Business 2005® 21 Biosensors – first generation H 2 O 2 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 H 2 O 2 usually by oxidation

22 Created with MindGenius Business 2005® 22 Biosensors – first generation First generation all involve five steps in the reaction: i. Diffusion from solution ii. Membrane transport iii. Reaction of enzyme with substrate iv. Reaction of enzyme with mediator (O 2 (aq) in this case) v. Reaction of mediator with the electrode

23 Created with MindGenius Business 2005® 23 Biosensors – first generation 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

24 Created with MindGenius Business 2005® 24 Biosensors – first generation Ideally, linear range should coincide with expected analyte concentrations Current is rate  i/nFA is flux in Fick’s Law Can extend linear range by using more enzyme, more active enzyme or decreasing membrane permeability Can extend linear range by using more enzyme, more active enzyme or decreasing membrane permeability

25 Created with MindGenius Business 2005® 25 Biosensors – first generation Deficiencies: Dependance on [O 2 (aq)] at low [O 2 (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

26 Created with MindGenius Business 2005® 26 Biosensors – second generation Other mediator species were investigated: Ferrocene mediated systems Quinone/dihydroquinone mediated systems Generally, Where M is a mediator

27 Created with MindGenius Business 2005® 27 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

28 Created with MindGenius Business 2005® 28 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

29 Created with MindGenius Business 2005® 29 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

30 Created with MindGenius Business 2005® 30 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

31 Created with MindGenius Business 2005® 31 Biosensors – inhibition based sensors “Chemical canaries” Will only give a qualitative YES/NO response “concentration devices” have LoDs ~ >  mol dm -3 Rate determining step is substrate diffusion

32 Created with MindGenius Business 2005® 32 Biosensors – inhibition based sensors “Chemical canaries” Enzyme turnover numbers are typically in the range 10 3 – 10 5 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

33 Created with MindGenius Business 2005® 33 Biosensors – inhibition based sensors “Chemical canaries” Example: CN - sensitive chemical canary Current used to regenerate Fc +  Fc is recorded

34 Created with MindGenius Business 2005® 34 Biosensors – inhibition based sensors “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

35 Created with MindGenius Business 2005® 35 Biosensors – inhibition based sensors “Chemical canaries” Example: Hydrogen Sulfide, H 2 S

36 Created with MindGenius Business 2005® 36 Biosensors – inhibition based sensors “Chemical canaries” Cyt OD is inhibited by H 2 S Reversible inhibition so H 2 S can be washed off Similar devices exist for organophosphates (nerve gases, insecticides) using acetylcholinesterase and chemical reactions coupled to this


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