Introduction to Electrochemical Biosensors, Lecture notes of Biomedicine

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Introduction to

Electrochemical Biosensors

Lecture 3

Summary

Introduction to Electrochemical Biosensors

‣ Potentiometric sensors ‣ Amperometric sensors ‣ Oxygen sensing (not a biosensor) ‣ Microelectrodes ‣ Glucose sensing

Electrochemical Sensors

Three main types of sensor

‣ Potentiometric:

• Use Ion Selective Electrodes to determine the concentration of chosen ions. ‣ Amperometric:

Measure current resulting from redox reactions. ‣ Conductometric

Measure changes in ionic composition resulting from an enzyme reaction.

Reference (Counter) Electrode E

Control or

Measurement

I

Electrochemical Sensors

• Potentiometric: Measure equilibrium E ( I = 0)
• Amperometric: Control E , measure I Working (Indicator, Detector) Electrode

Potentiometric Sensor

• pH - Common potentio- metric measurement
• Glass pH Electrode measures concentration of Hydronium (H

) ions

• This, and other potentio- metric sensors require a stable reference electrode E

Porous Frit Glass H

Membrane Sensing Electrode (Ag-AgCl) Reference Electrode (Ag-AgCl)

Potentiometric Biosensor

E

• Potentiometric urea sensor
• Two pH sensors: ‣ Sensing electrode coated with urease enzyme ‣ Bare reference sensor
• Local pH change at urease coated electrode
• pH (potential) difference between electrodes proportional to urea conc. Reference pH sensor Urease

Reference (Counter) Electrode E

Control or

Measurement

I

Electrochemical Sensors

• Potentiometric: Measure equilibrium E ( I = 0)
• Amperometric: Control E , measure I Working (Indicator, Detector) Electrode

Amperometric Oxygen Sensor

• Clark Oxygen Electrode
• Oxygen reduced at Pt cathode ring
• Anode coil reference electrode: Ag-AgCl
• Teflon membrane allows O 2 diffusion
• Current will depend on reaction rate and pO 2 ©Gary Christian, Analytical Chemistry, 6th Ed. (Wiley)

Electrode Surface Reactions

Flux of electrons Flux of Ox Flux of R Flux Balance at the Electrode Surface Planar Diffusion Stirring the solution reduces ‘Diffusion Barrier’ in Mass Transfer.

Hemispherical Diffusion

Electrode Surface Reactions

Flux of electrons Flux of Ox Flux of R Flux Balance at the Electrode Surface Microelectrodes in miniaturised sensors do not require stirring

Amperometric Glucose Sensor

• Glucose-oxidaze (GOx/ GOD) enzyme reaction
• How to measure this?
• Measure production of hydrogen peroxide?
• Electrochemical detection: H 2 O 2 → O 2 + 2H

• 2e

• Plateau current depends on H 2 O 2 conc. which depends on glucose conc. 0

Anodic

Cathodic

• i

• i

+ 0.6 V E

“First Generation” Sensor Issues

H 2 O 2 detection may be confounded with

interferant compounds such as uric acid.

Use of oxidases means that oxygen is

required, so the reaction is dependent on

this concentration.

How have these issues been addressed?

Reactions in GOx

• Flavin adenine dinucleotide (FAD) at the heart of GOx
• Redox active site: ‣ FAD - Oxidised form ‣ FADH^2 - Reduced form
• Interacting directly with the FAD could replace oxygen
• Difficult because it is buried away within the molecule GOx(FAD) + glucose → GOx(FADH 2 ) + glucono-lactone GOx(FADH 2 ) + O 2 → GOx(FAD) + H 2 O 2

“Second Generation” Sensors

Oxygen is replaced by mediator (Med):

GOx(FAD) + glucose + H 2 O → GOx(FADH 2 ) + glucono-lactone GOx(FADH 2 ) + 2Med(Ox) → GOx(FAD) + 2Med(Red) + 2H

• Mediator is oxidised at electrode to transfer electrons: GOx(FADH 2 ) + 2Med(Ox) → GOx(FAD) + 2Med(Red) + 2H

Common mediators include ferrocene,

ferricyanide, quinones and phenothiazone